Use of R-enantiomer of N-propargyl-1-aminoindan, salts, compositions and uses thereof

ABSTRACT

The subject invention provides methods of treating a subject afflicted with Parkinson&#39;s disease, memory disorder, depression, hyperactive syndrome, Attention Deficit Disorder, dementia, brain ischemia, stroke, head trauma injury, spinal trauma injury, neurotrauma, neurodegenerative disease, neurotoxic injury, multiple sclerosis, nerve damage, affective illness, schizophrenia or symptoms of withdrawal from an addictive substance, using the mesylate salt of R(+)-N-propargyl-1-aminoindan.

BACKGROUND OF THE INVENTION

I. The subject invention is in the field of selective irreversibleinhibitors of the enzyme monoamine oxidase (hereinafter MAO) andprovides the R(+) enantiomer of N-propargyl-1-aminoindan (also referredto herein as PAI) which is a selective irreversible inhibitor of theB-form of monoamine oxidase enzyme (hereinafter MAO-B). The subjectinvention also provides pharmaceutical compositions containing R(+)PAIwhich are particularly useful for the treatment of Parkinson's disease,a memory disorder, dementia, depression, hyperactive syndrome, anaffective illness, a neurodegenerative disease, a neurotoxic injury,stroke, brain ischemia, a head trauma injury, a spinal trauma injury,neurotrauma, schizophrenia, an attention deficit disorder, multiplesclerosis, and withdrawal symptoms.

II. Parkinson's disease is widely considered to be the result ofdegradation of the pre-synaptic dopaminergic neurons in the brain, witha subsequent decrease in the amount of the neurotransmitter dopaminebeing released. Inadequate dopamine release, therefore, leads to theonset of disturbances of voluntary muscle control, which disturbancesare symptomatic of Parkinson's disease.

Various methods of treating Parkinson's disease have been establishedand are currently in widespread use, including, for example, theadministration of L-DOPA together with a decarboxylase inhibitor such asL-carbidopa or benserazide. The decarboxylase inhibitor protects theL-DOPA molecule from peripheral decarboxylation and thus ensures L-DOPAuptake by the remaining dopaminergic neurons in the striatum of thebrain. Here, the L-DOPA is converted into dopamine resulting inincreased levels of dopamine in these neurons. In response tophysiological impulses, these neurons are therefore capable of releasinglarger amounts of dopamine at levels which approximate the normalrequired levels. L-DOPA treatment thus alleviates the symptoms of thedisease and contributes to the well-being of the patient.

However, L-DOPA treatment has its drawbacks, the main one being that itseffectiveness is optimal only during the first few years of treatment.After this period, the clinical response diminishes and is accompaniedby adverse side effects which include dyskinesia, fluctuation inefficacy throughout the day (“on-off effect”) and psychiatric symptomssuch as confusional states, paranoia, and hallucinations. This decreasein the effect of L-DOPA treatment is attributed to a number of factors,including the natural progression of the disease, alteration in dopaminereceptors as a consequence of increased dopamine production or increasedlevels of dopamine metabolites, and pharmacokinetic problems of L-DOPAabsorption (reviewed by Youdim, et al., Progress in Medicinal Chemistry,21, 138-167 (1984)).

In order to overcome the drawbacks of L-DOPA treatment, varioustreatments have been devised in which L-DOPA is combined with MAOinhibitors with the aim of reducing the metabolic breakdown of newlyformed dopamine (see for example, Chiese, P., U.S. Pat. No. 4,826,875,issued May 2, 1989).

MAO exists in two forms known as MAO-A and MAO-B which are selective fordifferent substrates and inhibitors. For example, MAO-B more efficientlymetabolizes substrates such as 2-phenylethylamine, and is selectivelyand irreversibly inhibited by (−)-deprenyl as described below.

It should be noted, however, that treatments combining L-DOPA with aninhibitor of both MAO-A and MAO-B are undesirable, as they lead toadverse side effects related to an increased level of catecholaminesthroughout the neuraxis. Furthermore, complete inhibition of MAO is alsoundesirable as it potentates the action of sympathomimetic amines suchas tyramine, leading to the so-called “cheese effect” (reviewed byYoudim et al., Handbook of Experimental Pharmacology, ed. byTrendelenburg and Weiner, Springer-Verlag, 90, ch. 3 (1988)). As MAO-Bwas shown to be the predominant form of MAO in the brain, selectiveinhibitors for this form are thus considered to be a possible tool forachieving a decrease in dopamine breakdown on the one hand, togetherwith a minimization of the systemic effects of total MAO inhibition onthe other.

Many inhibitors of MAO are chiral molecules. Although one enantiomeroften shows some stereoselectivity in relative potency towards MAO-A and-B, a given enantiomeric configuration is not always more selective thanits mirror image isomer in discriminating between MAO-A and MAO-B.

Table I lists the IC₅₀ (mmol/L) of enantiomeric pairs of propargylamines in a rat brain preparation of MAO. These results show smalldifferences in potency in MAO-B inhibition between the R and Senantiomers. (B. Hazelhoff, et al., Naunyn-Schmeideberg's Arch.Pharmacol., 330, 50 (1985)). Both enantiomers are selective for MAO-B.In 1967, Magyar, et al. reported that R-(−)-deprenyl is 500 times morepotent than the S-(+) enantiomer in inhibiting the oxidative deaminationof tyramine by rat brain homogenate. (K. Magyar, et al., Act. Physiol.Acad. Sci., Hung., 32, 377 (1967)).

In rat liver homogenate, R-deprenyl is only 15 times as potent as the Senantiomer. In other pharmacological activity assays, such as for theinhibition of tyramine uptake, deprenyl shows differentstereoselectivities. The S form is in certain cases the more potentepimer. (J. Knoll and K. Magyar, Advances in BiochemicalPsychopharmacology, 5, 393 (1972)).

N-Methyl-N-propargyl-1-aminotetralin (2-MPAT) is a close structuralanalogue of deprenyl. The absolute stereo-chemistry of 2-MPAT has notbeen assigned. However, the (+) isomer is selective for MAO-B and the(−) isomer is selective for MAO-A. The difference in potency between the2-MPAT enantiomers is less than 5-fold. (B. Hazelhoff, et al., id.). Theenantiomers of N-propargyl-1-aminotetralin (1-PAT) are also similar inactivity. The lack of data in Table I showing clear structure-activityrelationships between isolated (+) or (−)-2-MPAT makes it impossible topredict the absolute stereochemistry thereof.

After extensive computer modeling, Polymeropoulos recently predictedthat (R)-N-methyl-N-propargyl-1-aminoindan (R-1-MPAI) would be morepotent than (S) as a MAO-B inhibitor. (E. Polymeropoulos, Inhibitors ofMonoamine Oxidase B, I. Szelenyi, ed., Birkhauser Verlag, p. 110(1993)). However, experiments described show that R-1-MPAI is a slightlymore potent inhibitor of MAO-B than S-1-MPAI, but is an even more potentinhibitor of MAO-A. Both the selectivity between MAO-A and -B and therelative potency of the R and S epimers are low. Thus, contrary toexpectations in the art, 1-MPAI is useless as a pharmaceutical agent.

The data presented below demonstrate that high selectivity for MAO ofone enantiomer versus the other cannot be predicted. The structure ofthe MAO active site is not well enough understood to permit theprediction of the relative potency or selectivity of any given compoundor pair of enantiomers thereof.

III. Brain stroke is the third leading cause of death in the developedcountries. Survivors often suffer from neurological and motordisabilities. The majority of CNS strokes are regarded as localizedtissue anemia following obstruction of arterial blood flow which causesoxygen and glucose deprivation. Occlusion of the middle cerebral arteryin the rat (MCAO) is a common experimental procedure that is assumed torepresent stroke in humans. It has been proposed that the neurologicallesion caused by proximal occlusion of this artery in the ratcorresponds to a large focal cerebral infarct in humans (Yamori et al.,1976). This correspondence has been based on similarities betweencranial circulation in the two species. Other animal models of strokehave been described by Stefanovich (1983).

The histological changes described by Tamura et al. (1981) who were thefirst to introduce the MCAO procedure, were commonly seen in the cortexof the frontal (100%), sensimotor (75%) and auditory (75%) areas and toa lesser extent in the occipital lobe cortex (25%). In addition, damagewas observed in the lateral segment of the caudate nucleus (100%), andonly to a variable extent in its medial portion (38%). Concomitantly,the following disorders were reported in MCAO animals: neurologicaldeficits (Menzies et al., 1992), cognitive disturbances (Yamamoto etal., 1988), brain edema (Young et al., 1993; Matsui et al., 1993; Saur;et al., 1993), decreased cerebral blood flow (Teasdale et al., 1983),catecholamine fluctuations. (Cechetto et al., 1989). Any of thesedisorders might be indicative of the severity and extent of brain damagethat follow MCAO in the rat. Conversely, a drug with a potential tolimit or abort a given disorder may be considered as a candidate for thetreatment of stroke in humans. TABLE IA IC₅₀ (mmol/L) Data for Rat BrainMAO Inhibition by Propargylamines RELATIVE INHIBITION POTENCY COMPOUNDREF EPIMER A B A/B +/− 2-MPAI a + 140 16 8.8 A B − 46 88 0.5 3 0.2 R/SDEPRENYL a S 3600 16 120 80 2.6 R 450 6 75 1-MPAI b S 70 50 1.4 23 5 R 310 0.3 1-PAT c S 3800 50 76 4 0.5 R 900 90 10a. B. Hazelhoff, et al., Naunyn-Schmeideberg's Arch. Pharmacol., 330, 50(1985).b. European Patent Application 436,492 A2, published Jul. 10, 1991.c. Present inventors.

One selective MAO-B inhibitor, (−)-deprenyl, has been extensivelystudied and used as a MAO-B inhibitor to augment L-DOPA treatment. Thistreatment with (−)-deprenyl is generally favorable and does not causethe “cheese effect” at doses causing nearly complete inhibition of MAO-B(Elsworth, et al., Psychopharmacology, 57, 33 (1978)). Furthermore, theaddition of (−)-deprenyl to a combination of L-DOPA and a decarboxylaseinhibitor administered to Parkinsons's patients leads to improvements inakinesia and overall functional capacity, as well as the elimination of“on-off” type fluctuations (reviewed by Birkmayer & Riederer in“Parkinson's Disease,” Springer-Verlag, pp. 138-149 (1983)). Thus,(−)-deprenyl (a) enhances and prolongs the effect of L-DOPA, and (b)does not increase the adverse effects of L-DOPA treatment.

However, (−)-deprenyl is not without its own adverse sides effects,which include activation of pre-existing gastric ulcers and occasionalhypertensive episodes. Furthermore, (−)-deprenyl is an amphetaminederivative and is metabolized to amphetamine and methamphetamines, whichsubstances may lead to undesirable side effects such as increased heartrate (Simpson, Biochemical Pharmacology, 27, 1951 (1978); Finberg, etal., in “Monoamine Oxidase Inhibitors—The State of the Art,” Youdim andPaykel, eds., Wiley, pp. 31-43 (1981)).

Other compounds have been described that are selective irreversibleinhibitors of MAO-B but which are free of the undesirable effectsassociated with (−)-deprenyl. One such compound, namelyN-propargyl-1-aminoindan.HCl (racemic PAI.HCl), was described in GB1,003,666 and GB 1,037,014 and U.S. Pat. No. 3,513,244, issued May 19,1970. Racemic PAI.HCl is a potent, selective, irreversible inhibitor ofMAO-B, is not metabolized to amphetamines, and does not give rise tounwanted sympathomimetic effects.

In comparative animal tests, racemic PAI was shown to have considerableadvantages over (−)-deprenyl. For example, racemic PAI produces nosignificant tachycardia, does not increase blood pressure (effectsproduced by doses of 5 mg/kg of (−)-deprenyl), and does not lead tocontraction of nictitating membrane or to an increase in heart rate atdoses of up to 5 mg/kg (effects caused by (−)-deprenyl at doses over 0.5mg/kg). Furthermore, racemic PAI.HCl does not potentiate thecardiovascular effects of tyramine (Finberg, et al., in “Enzymes andNeurotransmitters in Mental Disease,” pp. 205-219 (1980), Usdin, et al.,Eds., Wiley, N.Y.; Finberg, et al. (1981), in “Monoamine OxidaseInhibitors—The State of the Art,” ibid.; Finberg and Youdim, BritishJournal Pharmacol., 85, 451 (1985)).

One underlying object of this invention was to separate the racemic PAIcompounds and to obtain an enantiomer with MAO-B inhibition activitywhich would be free of any undesirable side effects associated with theother enantiomer.

Since deprenyl has a similar structure to PAI and it is known that the(−)-enantiomer of deprenyl, i.e. (−)-deprenyl, is considerably morepharmaceutically active than the (+)-enantiomer, the (−) enantiomer ofPAI would be expected to be the more active MAO-B inhibitor.

However, contrary to such expectations, upon resolution of theenantiomers, it was found that the (+)-PAI enantiomer is in fact theactive MAO-B inhibitor while the (−)-enantiomer shows extremely lowMAO-B inhibitory activity. Furthermore, the (+)-PAI enantiomer also hasa degree of selectivity for MAO-B inhibition surprisingly higher thanthat of the corresponding racemic form, and should thus have fewerundesirable side effects in the treatment of the indicated diseases thanwould the racemic mixture. These findings are based on both in vitro andin vivo experiments as discussed in greater detail infra.

It was subsequently shown that (+)-PAI has the R absolut

configuration. This finding was also surprising based o

the expected structural similarity of (+)-PAI analogy wit

deprenyl and the amphetamines.

The high degree of stereoselectivity of pharmacologica

activity between R(+)-PAI and the S(−) enantiomer a

discussed hereinbelow is also remarkable. The compoun

R(+)-PAI is nearly four orders of magnitude more active tha

the S(−) enantiomer in MAO-B inhibition. This ratio i

significantly higher than that observed between the tw

deprenyl enantiomers (Knoll and Magyar, Adv. Biochem

Psychopharmacol., 5, 393 (1972); Magyar, et al., Act

Physiol. Acad. Sci. Hung., 32, 377 (1967)). Furthermore, i

some physiological tests, (+)-deprenyl was reported to hav

activity equal to or even higher than that of the (−

enantiomer (Tekes, et al., Pol. J. Pharmacol. Pharm., 40

653 (1988)).

MPAI is a more potent inhibitor of MAO activity, but wit

lower selectivity for MAO-B over A (Tipton, et al., Biochen

Pharmacol., 31, 1250 (1982)). As only a small degree c

difference in the relative activities of the two resolve

enantiomers was surprisingly observed with MPAI, t

remarkable behavior of R(+)PAI is further emphasized (S

Table 1B).

The subject invention also provides methods of using t

pharmaceutically active PAI-enantiomer alone (without

DOPA) for treatment of Parkinson's disease, a memo

disorder, dementia, depression, hyperactive syndrome,

affective illness, a neurodegenerative disease, a neurotox

injury, brain ischemia, a head trauma injury, a spin

trauma injury, schizophrenia, an attention deficit disorde

multiple sclerosis, or withdrawal symptoms (see review

Youdim, et al., in Handbook of Experimental Pharmacology, Trendelenbergand Wiener, eds., 90/I, ch. 3 (1988)).

The subject invention further provides a method of using thepharmaceutically active PAI-enantiomer alone for pre-treatment ofParkinson's disease. The subject invention also provides pharmaceuticalcompositions comprising R(+)PAI and synergistic agents such as levodopa.The use of such agents has been studied with respect to (−)-deprenylwhich was shown to be effective when administered alone to earlyParkinson's patients, and may also have a synergistic effect in thesepatients when administered together with α-tocopherol, a vitamin Ederivative (The Parkinson's Study Group, New England J. Med., 321(20),1364-1371 (1989)).

In addition to its usefulness in treating Parkinson's disease,(−)-deprenyl has also been shown to be useful in the treatment ofpatients with dementia of the Alzheimer type (DAT) (Tariot, et al.,Psychopharmacology, 91, 489-495 (1987)), and in the treatment ofdepression (Mendelewicz and Youdim, Brit. J. Psychiat. 142, 508-511(1983)). The R(+)PAI compound of this invention, and particularly themesylate salt thereof, has been shown to restore memory. R(+)PAI thushas potential for the treatment of memory disorders, dementia,especially of the Alzheimer's type, and hyperactive syndrome inchildren.

Finally, the subject invention provides highly stable salts of R(+)PAIwith superior pharmaceutical properties. The mesylate salt is especiallystable, shows unexpectedly greater selectivity, and shows significantlyfewer side effects than do the corresponding racemic salts.

SUMMARY OF THE INVENTION

The subject invention provides R(+)-N-propargyl-1-aminoindan having thestructure:

The subject invention further provides a pharmaceutically acceptablesalt of R(+)-N-propargyl-1-aminoindan.

The subject invention further provides a pharmaceutical compositionwhich comprises a therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier.

The subject invention further provides a method of treating a subjectafflicted with Parkinson's disease which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat Parkinson's disease in the subject.

The subject invention further provides a method of treating a subjectafflicted with a memory disorder which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the memory disorder in the subject.

The subject invention further provides a method of treating a subjectafflicted with dementia which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to treatdementia in the subject. In one embodiment, the dementia is of theAlzheimer type (DAT).

The subject invention further provides a method of treating a subjectafflicted with depression which comprises administering to the subjectan amount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to treatdepression in the subject.

The subject invention further provides a method of treating a subjectafflicted with hyperactive syndrome which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat hyperactive syndrome in the subject.

The subject invention further provides a method of treating a subjectafflicted with an affective illness which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the affective illness in the subject.

The subject invention further provides a method of treating a subjectafflicted with a neurodegenerative disease which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurodegenerative disease in the subject.

The subject invention further provides a method of treating a subjectafflicted with a neurotoxic injury which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurotoxic injury in the subject.

The subject invention further provides a method of treating a subjectafflicted with brain ischemia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat brain ischemia in the subject.

The subject invention further provides a method of treating a subjectafflicted with a head trauma injury which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the head trauma injury in the subject.

The subject invention further provides a method of treating a subjectafflicted with a spinal trauma injury which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the spinal trauma injury in the subject.

The subject invention further provides a method of treating a subjectafflicted with schizophrenia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat schizophrenia in the subject.

The subject invention further provides a method of treating a subjectafflicted with an attention deficit disorder which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the attention deficit disorder in the subject.

The subject invention further provides a method of treating a subjectafflicted with multiple sclerosis which comprises administering to thesubject an amount of R(+)-N-propargyl-3-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat multiple sclerosis in the subject.

The subject invention further provides a method of preventing nervedamage in a subject which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to preventnerve damage in the subject.

The subject invention further provides a method of treating a subjectsuffering from symptoms of withdrawal from an addictive substance whichcomprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable saltthereof of the subject invention effective to treat the symptoms ofwithdrawal in the subject.

The subject invention further provides a method for preparingR(+)-N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, R(−)-aminoindan with eitherpropargyl bromide or propargyl chloride so as to formR(+)-N-propargyl-1-aminoindan, and isolating theR(+)-N-propargyl-1-aminoindan formed thereby.

The subject invention further provides a method for preparing racemicN-propargyl-1-aminoindan which comprises contacting, in the presence ofan organic or inorganic base, racemic 1-aminoindan with propargylbromide or propargyl chloride so as to form racemicN-propargyl-1-aminoindan, and isolating the racemicN-propargyl-1-aminoindan formed thereby.

Finally, the subject invention provides a method of preparing anR(+)-N-propargyl-1-aminoindan salt which comprises contacting racemicN-propargyl-1-aminoindan with an optically active acid so as to form twodiastereomeric N-propargyl-1-aminoindan salts, and isolatingR(+)-N-propargyl-1-aminoindan salt from the diastereomericN-propargyl-1-aminoindan salts so formed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of the results according to Example22 showing in vitro MAO-A inhibitory activity.

FIG. 2 is a graphic representation of the results according to Example22 showing in vitro MAO-B inhibitory activity.

FIG. 3 is a graphic representation of the results according to Example22 showing MAO activity in human cortical tissue.

FIG. 4 is a graphic representation of the results according to Example23 showing acute inhibition (i.p.) of MAO-A in brain.

FIG. 5 is a graphic representation of the results according to Example23 showing acute inhibition (i.p.) of MAO-B in brain.

FIG. 6 is a graphic representation of the results according to Example23 showing acute inhibition (i.p.) of MAO-A in liver.

FIG. 7 is a graphic representation of the results according to Example23 showing acute inhibition (i.p.) of MAO-B in liver.

FIG. 8 is a graphic representation of the results according to Example23 showing acute inhibition (per os) of MAO-A in brain.

FIG. 9 is a graphic representation of the results according to Example23 showing acute inhibition (per os) of MAO-B in brain.

FIG. 10 is a graphic representation of the results according to Example23 showing acute inhibition (per os) of MAO-A in liver.

FIG. 11 is a graphic representation of the results according to Example23 showing acute inhibition (per os) of MAO-B in liver.

FIG. 12 is a graphic representation of the results according to Example24 showing chronic inhibition of MAO-A in brain.

FIG. 13 is a graphic representation of the results according to Example24 showing chronic inhibition of MAO-B in brain.

FIG. 14 is a graphic representation of the results according to Example24 showing chronic inhibition of MAO-A in liver.

FIG. 15 is a graphic representation of the results according to Example24 showing chronic inhibition of MAO-B in liver.

FIG. 16 is a graphic representation of the results according to Example25 showing MAO-B activity in rat brain as a function of time followingi.p. administration of R(+)PAI.

FIG. 17 is a graphic representation of the results according to Example32 showing restoration of normokinesia in mice that had receivedhaloperidol 6 mg/kg s.c. Mice received each of the test drugs i.p. atthe indicated dose. 2 hours later they received haloperidol. Kineticscores were taken 3 hours after haloperidol. These scores consisted ofthe ability to move horizontally along a rod, the ability to descend avertical rod, and the shortening of catalepsia. In the absence ofhaloperidol, the maximum score is 12, with haloperidol alone, 6.6±0.03.Statistical significance was calculated by the Student's “t” test: *p≦0.05; ** p≦0.01; * * *p≦0.001 with respect to haloperidol alone. Thescores of (R)-PAI are significantly different from those of racemic-PAIat 5 mg/kg (p≦0.05), at 10 mg/kg (p≦0.01), and at 15 mg/kg (p≦0.05),(n=5.6). The dosage shown is for the free base of PAI (and not themesylate salt).

FIG. 18 is a graphic representation of the results according to Example32 showing restoration of motor activity in rats treated withα-methyl-p-tyrosine at 100 mg/kg i.p. Rats received the test drug i.p.at the indicated doses. After two hours they received α-Mpt and wereimmediately placed in activity cages. Total motor activity was recordedfor the duration of 10 hours. Control rats, treated with saline, onlyscored 15, 862+1424. With α-Mpt alone, they scored 8, 108±810.Statistical significance by the Student's “t” test: *p≦0.05; **p≦0.01;***p≦0.001 with respect to α-MpT alone. The scores of (R)-PAI aresignificantly different from racemic-PAI at 2 mg/kg (p≦0.01), (n=6).Dosage shown is for the free base of PAI and not the mesylate salt.

FIG. 19 is a graph showing the NADH response to 2 minutes of anoxiameasured 30 minutes after injury and at half-hour intervals thereafter.

FIG. 20 Ischemic brain lesion evaluation with MRI T2-scan 48 hours afterMCA-O and [R] (+)PAI Mesylate Treatment in rats: The middle cerebralartery was surgically occluded as described in Example 38. [R](+)PAIMesylate was administered as follows: 1.0 mg/kg ip immediately aftersurgery; 0.5 mg/kg ip, 2 hrs after surgery; 1.0 mg/kg ip, 24 hrs aftersurgery. Infarct volume (mm³) was determined by MRI 48 hoursfollowing-surgery.

FIG. 21: neurological evaluation of Wistar rats subjected to MCA-O and[R](+)PAI Mesylate Treatment: The middle cerebral artery was surgicallyoccluded and [R](+)PAI Mesylate administered as in FIG. 20. At 24 hourspost surgery a neurological score was taken as described in Example 38.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides R(+)-N-propargyl-1-aminoindan having thestructure:

As demonstrated in the Experimental Examples hereinbelow, R(+)PAI isnearly 7,000 times more active as an inhibitor of MAO-B than is S(−)PAI.In view of known MAO-B inhibitors in the art which possess lowselectivity between MAO-A and MAO-B, and which do not show predictabletrends in potency as a function of R or S configuration, the selectivityof R(+)PAI is unexpected.

R(+)PAI may be obtained by optical resolution of racemic mixtures of R-and S-enantiomers of PAI. Such a resolution can be accomplished by anyconventional resolution method well known to a person skilled in theart, such as those described in J. Jacques, A. Collet and S. Wilen,“Enantiomers, Racemates and Resolutions,” Wiley, N.Y. (1981). Forexample, the resolution may be carried out by preparative chromatographyon a chiral column. Another example of a suitable resolution method isthe formation of diastereomeric salts with a chiral acid such astartaric, malic, mandelic acid or N-acetyl derivatives of amino acids,such as N-acetyl leucine, followed by recrystallisation to isolate thediastereomeric salt of the desired R enantiomer.

The racemic mixture of R and S enantiomers of PAI may be prepared, forexample, as described in GB 1,003,676 and GB 1,037,014. The racemicmixture of PAI can also be prepared by reacting 1-chloroindan withpropargylamine. Alternatively, this racemate may be prepared by reactingpropargylamine with 1-indanone to form the corresponding imine, followedby reduction of the carbon-nitrogen double bond of the imine with asuitable agent, such as sodium borohydride.

In accordance with this invention, the R enantiomer of PAI can also beprepared directly from the optically active R-enantiomer of 1-aminoindanby reaction with propargyl bromide or propargyl chloride in the presenceof an organic or inorganic base, and optionally in the presence of asuitable solvent.

Suitable organic or inorganic bases for use in the above reactioninclude, by way of example, triethylamine, pyridine, alkali metalcarbonates, and bicarbonates. If the reaction is conducted in thepresence of a solvent, the solvent may be chosen from, e.g., toluene,methylene chloride, and acetonitrile. One method of preparing R(+)PAI isto react R-1-aminoindan with propargyl chloride using potassiumbicarbonate as a base and acetonitrile as solvent.

The above-described reaction of 1-aminoindan generally results in amixture of unreacted primary amine, the desired secondary amine and thetertiary amine N,N-bispropargylamino product. The desired secondaryamine, i.e., N-propargyl-1-aminoindan, can be separated from thismixture by a conventional separation method including, by way ofexample, chromatography, distillation and selective extraction.

The R-1-aminoindan starting material can be prepared by methods known inthe art which include, by way of example, the method of Lawson and Rao,Biochemistry, 19, 2133 (1980), methods in references cited therein, andthe method of European Patent No. 235,590.

R-1-aminoindan can also be prepared by resolution of a racemic mixtureof the R and S enantiomers, which involves, for example, the formationof diastereomeric salts with chiral acids, or any other known methodsuch as those reported in J. Jacques, et al., ibid. Alternatively,R-1-aminoindan may be prepared by reacting 1-indanone with an opticallyactive amine, followed by reduction of the carbon nitrogen double bondof the resulting imine by hydrogenation over a suitable catalyst, suchas palladium on carbon, platinum oxide or Raney nickel. Suitableoptically active amines include, for example, one of the antipodes ofphenethylamine or an ester of an amino acid, such as valine orphenylalanine. The benzylic N—C bond may be cleaved subsequently byhydrogenation under non-vigorous conditions.

An additional method for preparing R-1-aminoindan is the hydrogenationof indan-1-one oxime ethers as described above, wherein the alkylportion of the ether contains an optically pure chiral center.Alternatively, a non-chiral derivative of indan-1-one containing acarbon-nitrogen double bond, such as an imine or oxime, can be reducedwith a chiral reducing agent, e.g., a complex of lithiumaluminum-hydride and ephedrine.

The subject invention further provides a pharmaceutically acceptablesalt of R(+)-N-propargyl-1-aminoindan.

In the practice of this invention, pharmaceutically acceptable saltsinclude, but are not limited to, the mesylate, maleate, fumarate,tartrate, hydrochloride, hydrobromide, esylate, p-toluenesulfonate,benzoate, acetate, phosphate and sulfate salts.

In one embodiment, the salt is selected from the group consisting of themesylate salt of R(+)-N-propargyl-1-aminoindan, the esylate salt ofR(+)-N-propargyl-1-aminoindan, and the sulfate salt ofR(+)-N-propargyl-1-aminoindan.

As demonstrated in the Experimental Examples hereinbelow, the mesylatesalt is highly stable to thermal degradation, and shows unexpectedlysuperior selectivity for MAO-B over the racemic salt.

For the preparation of pharmaceutically acceptable acid addition saltsof the compound of R(+)PAI, the free base can be reacted with thedesired acids in the presence of a suitable solvent by conventionalmethods. Similarly, an acid addition salt may be converted to the freebase form in a known manner.

A preferred mode of preparing the mesylate salt of (R)-PAI comprises (a)adding an aqueous solution of 15% sodium hydroxide to a solution ofpropargyl benzenesulfonate (or tosylate or mesylate) in toluene; (b)stirring for 5 hours; (c) adding additional toluene and water; (d)separating and washing the organic phase with 10% sodium hydroxide, andthen diluting with water; (e) adjusting the pH of the mixture to 3.2 byadding 10% aqueous sulfuric acid; (f) separating the aqueous phase andadjusting the pH to 7.3 with 10% sodium hydroxide; (g) extracting threetimes with toluene while maintaining constant pH; (h) concentratingcombined organic layers in vacuo to give a yellow oil; (i) dissolvingthe oil and L-tartaric acid in isopropanol; (j) heating to reflux for 1hour; (k) cooling to room temperature and collecting the precipitate byfiltration; (l) recrystallizing the crude di-propargylaminoindantartrate from methanol/isopropanol (1:1) to givedi(R(+)-N-propargyl-1-aminoindan) tartrate; (m) dissolving the tartratesalt and methanesulfonic acid in isopropanol, and heating to reflux for30 minutes; and (n) cooling to room temperature, and collecting theprecipitated R(+)-N-propargyl-1-aminoindan.

The subject invention further provides a pharmaceutical compositionwhich comprises a therapeutically effective amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier. The “therapeuticallyeffective amount” of the R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof may be determined according tomethods well known to those skilled in the art.

Possible salts useful for such compositions include hydrochloride,phosphate, maleate, fumarate, tartrate, mesylate, esylate, and sulfatesalts.

These compositions may be prepared as medicaments to be administeredorally, parenterally, rectally, or transdermally.

In one embodiment, the pharmaceutically acceptable carrier is a solidand the pharmaceutical composition is a tablet. The therapeuticallyeffective amount may be an amount from about 0.1 mg to about 100 mg. Thetherapeutically effective amount may also be an amount from about 1 mgto about 10 mg.

Suitable forms for oral administration include tablets, compressed orcoated pills, dragees, sachets, hard or soft gelatin capsules,sublingual tablets, syrups and suspensions.

In an alternative embodiment, the pharmaceutically acceptable carrier isa liquid and the pharmaceutical composition is an injectable solution.The therapeutically effective amount may be an amount from about 0.1mg/ml to about 100 mg/ml. The therapeutically effective amount may alsobe an amount from about 1 mg/ml to about 10 mg/ml. In one embodiment,the dose administered is an amount between 0.5 ml and 1.0 ml.

In a further alternative embodiment, the carrier is a gel and thepharmaceutical composition is a suppository.

For parenteral administration the invention provides ampoules or vialsthat include an aqueous or non-aqueous solution or emulsion. For rectaladministration there are provided suppositories with hydrophilic orhydrophobic vehicles. For topical application as ointments andtransdermal delivery there are provided suitable delivery systems asknown in the art.

In the preferred embodiment, the pharmaceutically acceptable salt is amesylate salt.

These compositions may be used alone to treat the above-listeddisorders, or alternatively, as in the case of Parkinson's disease, forexample, they may be used as an adjunct to the conventional L-DOPAtreatments.

The preferred dosages of the active ingredient, i.e., R-PAI, in theabove compositions are within the following ranges. For oral orsuppository formulations, 0.1-100 mg per dosage unit may be taken daily,and preferably 1-10 mg per dosage unit is taken daily. For injectableformulations, 0.1-100 mg/ml per dosage unit may be taken daily, andpreferably 1-10 mg/ml per dosage unit is taken daily.

In one embodiment, the pharmaceutical composition further comprises atherapeutically effective amount of Levodopa. In another embodiment, thepharmaceutical composition still further comprises an effective amountof a decarboxylase inhibitor.

The amount of decarboxylase inhibitor administered in combination with(R)-PAI or a pharmaceutically acceptable salt thereof is an amounteffective to ensure L-DOPA uptake in the subject.

The decarboxylase inhibitor may be L-Carbidopa. In one embodiment, thetherapeutically effective amount of R(+)-N-propargyl-1-aminoindan isabout 0.1 mg to about 100 mg, the therapeutically effective amount ofLevodopa is about 50 mg to about 250 mg, and the effective amount ofL-Carbidopa is about 10 mg to about 25 mg.

The decarboxylase inhibitor may also be benserazide. In one embodiment,the therapeutically effective amount of R(+)-N-propargyl-1-aminoindan isabout 0.1 mg to about 100 mg, the therapeutically effective amount ofLevodopa is about 50 mg to about 200 mg, and the effective amount ofbenserazide is about 12.5 mg to about 50 mg.

The subject invention further provides a method of treating a subjectafflicted with Parkinson's disease which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat Parkinson's disease in the subject.

Methods of treatment of Parkinson's disease which combine the use of(R)-PAI with other drugs, such as dopamine agonists, bromocryptine,pergolide, lisuride, as well as catecholamine oxidase methyl transferaseinhibitors are within the scope of the subject invention.

In the preferred embodiment, the pharmaceutically acceptable salt is amesylate salt.

The administering may comprise orally administering, rectallyadministering, transdermally administering, or parenterallyadministering.

In one embodiment, the method of the subject invention further comprisesadministering to the subject a therapeutically effective amount ofLevodopa. In another embodiment, the method of the subject inventionstill further comprises administering to the subject an effective amountof a decarboxylase inhibitor.

The decarboxylase inhibitor may be L-Carbidopa. Alternatively, thedecarboxylase inhibitor may be benserazide.

The subject invention further provides a method of treating a subjectafflicted with a memory disorder which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the memory disorder in the subject.

The subject invention further provides a method of treating a subjectafflicted with dementia which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to treatdementia in the subject. In one embodiment, the dementia is of theAlzheimer type (DAT).

The subject invention further provides a method of treating a subjectafflicted with depression which comprises administering to the subjectan amount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to treatdepression in the subject.

The subject invention further provides a method of treating a subjectafflicted with hyperactive syndrome which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat hyperactive syndrome in the subject.

The administering may comprise orally administering, rectallyadministering, or parenterally administering.

The subject invention further provides a method of treating a subjectafflicted with an affective illness which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the affective illness in the subject.

The subject invention further provides a method of treating a subjectafflicted with a neurodegenerative disease which comprises administeringto the subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurodegenerative disease in the subject.

The subject invention further provides a method of treating a subjectafflicted with a neurotoxic injury which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the neurotoxic injury in the subject.

The subject invention further provides a method of treating a subjectafflicted with brain ischemia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat brain ischemia in the subject.

This invention provides a method of treating brain ischemia or stroke ina subject which comprises administering to the subject an amount ofR(+)-N-propargyl-3-aminoindan or a pharmaceutically acceptable saltthereof effective to treat brain ischemia or stroke in the subject.

In an embodiment of the method for treatment of brain ischemia orstroke, the pharmaceutically acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt. Preferably, the pharmaceutically acceptable salt isthe mesylate salt of R(+)-N-propargyl-1-aminoindan.

The effective amount can be determined using techniques known to thoseof skill in the art, such as titration. In an embodiment of thisinvention, the effective amount is from about 0.5 milligrams perkilogram body weight of the subject to about 2.5 milligrams per kilogrambody weight of the subject. The R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof is administered usingtechniques known to those of skill in the art. For example, it may beadministered intravenously, orally, rectally, transdermally, orparenterally.

The subject is preferably a mammal, such as a dog, cat, mouse, rat,rabbit, pig, horse, goat, sheep, cow, ape or monkey. In a particularembodiment the subject is human.

In an embodiment of this invention, the effective amount is from about0.01 mg to 50.0 mg per day. In a more specific embodiment, the effectiveamount is from 0.1 to 10.0 mg per day.

In one embodiment of the above-described method, the area of the brainischemia is reduced by about thirty-five percent.

The subject invention further provides a method of treating a subjectafflicted with a head trauma injury which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the head trauma injury in the subject.

The subject invention further provides a method of treating a subjectafflicted with a spinal trauma injury which comprises administering tothe subject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat the spinal trauma injury in the subject.

This invention further provides a method of treating neurotrauma in asubject which comprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or a pharmaceutically acceptable saltthereof effective to treat neurotrauma in the subject.

In the treatment of head trauma injury, spinal trauma injury orneurotrauma, the pharmaceutically acceptable salt ofR(+)-N-propargyl-1-aminoindan is selected from the group consisting of:the mesylate salt; the ethylsulfonate salt; the sulfate salt; and thehydrochloride salt. Preferably, the pharmaceutically acceptable salt isthe mesylate salt of R(+)-N-propargyl-1-aminoindan.

The effective amount can be determined using techniques known to thoseof skill in the art, such as titration. In an embodiment of thisinvention, the effective amount is from about 0.5 milligrams perkilogram body weight of the subject to about 2.5 milligrams per kilogrambody weight of the subject. The R(+)-N-propargyl-1-aminoindan orpharmaceutically acceptable salt thereof is administered usingtechniques known to those of skill in the art. For example, it may beadministered intravenously, orally, rectally, transdermally, orparenterally.

The subject is preferably a mammal, such as a dog, cat, mouse, rat,rabbit, pig, horse, goat, sheep, cow, ape or monkey. In a particularembodiment the subject is human.

In an embodiment of this invention, the effective amount is from about0.01 mg to 50.0 mg per day. In a more specific embodiment, the effectiveamount is from 0.1 to 10.0 mg per day.

The subject invention further provides a method of treating a subjectafflicted with schizophrenia which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat schizophrenia in the subject.

The subject invention further provides a method of treating a subjectafflicted with an attention deficit disorder which comprisesadministering to the subject an amount of R(+)-N-propargyl-1-aminoindanor the pharmaceutically acceptable salt thereof of the subject inventioneffective to treat the attention deficit disorder in the subject.

The subject invention further provides a method of treating a subjectafflicted with multiple sclerosis which comprises administering to thesubject an amount of R(+)-N-propargyl-1-aminoindan or thepharmaceutically acceptable salt thereof of the subject inventioneffective to treat multiple sclerosis in the subject.

The subject invention further provides a method of preventing nervedamage in a subject which comprises administering to the subject anamount of R(+)-N-propargyl-1-aminoindan or the pharmaceuticallyacceptable salt thereof of the subject invention effective to preventnerve damage in the subject.

In one embodiment, the nerve damage is structural nerve damage. Inanother embodiment, the structural nerve damage is optic nerve damage.

The subject invention further provides a method of treating a subjectsuffering from symptoms of withdrawal from an addictive substance whichcomprises administering to the subject an amount ofR(+)-N-propargyl-1-aminoindan or the pharmaceutically acceptable saltthereof of the subject invention effective to treat the symptoms ofwithdrawal in the subject.

As used herein, the term “symptoms of withdrawal” refers to physicaland/or psychological symptoms, including drug craving, depression,irritability, anergia, amotivation, appetite change, nausea, shaking andsleep irregularity.

As used herein, the term “addictive substance” includes, by way ofexample, (a) addictive opiates such as opium, heroin and morphine, (b)psychostimulants such as cocaine, amphetamines and methamphetamines, (c)alcohol, (d) nicotine, (e) barbiturates and (f) narcotics such asfentanyl, codeine, diphenoxylate and thebaine.

In one embodiment, the addictive substance is cocaine. In anotherembodiment, the addictive substance is alcohol.

The subject invention further provides a method for preparingR(+)-N-propargyl-1-aminoindan which comprises contacting, in thepresence of an organic or inorganic base, R(−)-aminoindan with eitherpropargyl bromide or propargyl chloride so as to formR(+)-N-propargyl-1-aminoindan, and isolating theR(+)-N-propargyl-1-aminoindan formed thereby.

The subject invention further provides a method for preparing racemicN-propargyl-1-aminoindan which comprises contacting, in the presence ofan organic or inorganic base, racemic 1-aminoindan with propargylbromide or propargyl chloride so as to form racemicN-propargyl-1-aminoindan, and isolating the racemicN-propargyl-1-aminoindan formed thereby.

Finally, the subject invention provides a method of preparing anR(+)-N-propargyl-1-aminoindan salt which comprises contacting racemicN-propargyl-1-aminoindan with an optically active acid so as to form twodiastereomeric N-propargyl-1-aminoindan salts, and isolatingR(+)-N-propargyl-1-aminoindan salt from the diastereomericN-propargyl-1-aminoindan salts so formed.

In one embodiment, the isolating comprises isolating by fractionalcrystallization.

The following Experimental Details are set forth to aid in anunderstanding of the invention, and are not intended, and should not beconstrued, to limit in any way the invention set forth in the claimswhich follow thereafter.

Experimental Details

EXAMPLE 1 Racemic N-propargyl-1-aminoindan hydrochloride

10.0 g of racemic 1-aminoindan and 10.49 of potassium carbonate wereadded to 75 ml of acetonitrile. The resulting suspension was heated to60° C. and 4.5 g of propargyl chloride was added dropwise.

The mixture was stirred at 60° C. for 16 hours, whereafter most of thevolatiles were removed by distillation in vacuo. The residue waspartitioned between 10% aqueous sodium hydroxide and methylene chloride.

The organic phase was dried and the solvent removed by distillation. Theresidue was flash chromatographed on silica gel, eluting with 40%; ethylacetate/60% hexane. The fractions containing the title compound as afree base were combined and the eluant replaced by ether. The etherealsolution was treated with gaseous HCl, the precipitate formed wasisolated by suction filtration and recrystallized from isopropanol toyield 7.3 g of the title compound, m.p. 182-4° C.

Chromatographic and spectroscopic data were in accordance with U.S. Pat.No. 3,513,244, issued May 19, 1970, and an authentic sample and were asfollows: NMR δ (CDCl₃): 2.45 (2H, m), 2.60 (1H, t), 2.90 (1H, m), 3.45(1H, m), 3.70 (2H, d), 4.95 (1H, t), 7.5 (4H, m) ppm.

EXAMPLE 2 S-(−)-N-Propargyl-1-aminoindan hydrochloride

The title compound in free base form was isolated by resolving theracemic mixture of the free base of Example 1 on a Chiracel OJ(cellulose tris [p-methylbenzoate]) preparative HPLC column eluting with10% isopropanol/90% hexane and collecting the first eluted major peak.The resulting oil was converted to the title compound (hydrochloride) bytreatment of a 10% diethyl ether solution of the oil with gaseous HCl,and the resulting precipitate was collected by suction filtration.[a]_(D)−29.2° (1%, ethanol), m.p. 182-184° C. Other chromatographic andspectroscopic properties were identical with the hydrochloride salt ofExample 1.

EXAMPLE 3 R-(′)-N-Propargyl-1-aminoindan hydrochloride

The title compound was prepared as in Example 2 above, except that thesecond eluted peak from the preparative HPLC was collected:[a]_(D)+29.1° (0.8%, ethanol), m.p. 179-181° C. Other chromatographicand spectroscopic properties were identical with the hydrochloride saltof Example 1.

EXAMPLE 4 R-(+)-N-Propargyl-1-aminoindan hydrochloride

12.4 g of R-(−)-1-Aminoindan and 12.9 g of potassium carbonate wereadded to 95 ml of acetonitrile. The resulting suspension was heated to60° C. and 5.6 g of propargyl chloride was added dropwise. The mixturewas stirred at 60° C. for 16 hours, whereafter most of the volatileswere removed by distillation in vacuo. The residue was partitionedbetween 10% aqueous sodium hydroxide and methylene chloride.

The organic phase was dried and the solvent removed in vacuo. Theresidue was flash chromatographed on silica get eluting with 40% ethylacetate/60% hexane. Fractions containing the free base of the titlecompound were combined and the solvent replaced by ether. The etherealsolution was treated with gaseous HCl and the resulting precipitate wasisolated by suction filtration and recrystallized from isopropanol toyield 6.8 g of the title compound, m.p. 163-185° C., [a]_(D)+30.90 (2%ethanol). Spectral properties were identical to those reported for thecompound of Example 1.

EXAMPLE 5 S-(−)-N-Propargyl-1-aminoindan hydrochloride

The title compound was prepared by the method of Example 4, except thatS-(+)-1-aminoindan was used as starting material. The product exhibited[a]_(D)−30.3 (2% ethanol), m.p. 183-5° C. Spectral properties wereidentical to those reported for the compound of Example 1.

EXAMPLE 6A Di(R-(+)-N-propargyl-1-aminoindan) L-tartrate

To a solution of tartaric acid (4.4 g) in 48 ml of boiling methanol wasadded a solution of R-(+)-N-propargyl-1-aminoindan free base (5.0 g) inmethanol (48 ml). The solution was heated to reflux and 284 ml oft-butylmethyl ether was added over 20 minutes. The mixture was heatedfor an additional 30 minutes, cooled, and the resulting precipitate wasisolated by suction filtration to yield 6.7 g of the title compound:m.p. 175-177° C.; [α]_(D) (1.5, H₂O)=+34.3; Anal. calcd. for C₂₈H₃₂O₆N₂;C, 68.26, H, 6.56, N, 5.69. Found: C, 68.76; H, 6.57; N, 5.61.

EXAMPLE 6B R-(+)-N-propargyl-1-aminoindan mesylate

a) To a solution of propargyl benzenesulfonate (78.4 g) and racemicaminoindan (63.2 g) in toluene (240 mL) at 20° C. was added dropwise anaqueous solution of 15% sodium hydroxide (108 mL). After 5 hours ofstirring, additional toluene (80 mL) and water (200 mL) were added withstirring. The organic phase was separated and washed with 10% aqueoussodium hydroxide and then diluted with water. The pH of the mixture wasadjusted to 3.2 by the addition of 10% aqueous sulfuric acid. Theaqueous phase was separated and its pH was adjusted to 7.3 with 10%sodium hydroxide and extracted three times with toluene whilemaintaining constant pH. The combined organic layers were concentratedin vacuo to 40.7 g of a yellow oil.

b) The above crude racemic propargylaminoindan and L-tartaric acid (10g) were dissolved in isopropanol (1 L) and heated to reflux for 1 hour.The reaction was then allowed to cool to room temperature with stirringand the precipitate collected by filtration. The crudedi-propargylaminoindan tartrate was recrystallized from 1 L of 1:1methanol/isopropanol to givedi(R-(+)-N-propargyl-1-aminoindan)-L-tartrate with physical and spectralproperties identical to that of the compound of Example 6A.

c) A solution of di-(R-(+)-N-propargyl-1-aminoindan) tartrate (15 g) andmethanesulfonic acid (6 g) in isopropanol (150 mL) was heated to refluxfor 30 minutes The reaction was allowed to cool to room temperature andthe resulting precipitate isolated by suction filtration to give thetitle compound (11.1 g) with m.p. 157° C. and [α]_(D)=22°.

EXAMPLE 7 R-(+)-N-Methyl-N-propargyl-1-aminoindan hydrochloride

The free base form of R-(+)-N-propargyl-1-aminoindan from Example 4 (1.2grams), potassium carbonate (0.97 grams) and methyl iodide (1 gram) wereadded to 15 ml of acetone and the resulting suspension heated to refluxunder a nitrogen atmosphere for 8 hours. Thereafter the volatiles wereremoved under reduced pressure and the residue partitioned between 10%aqueous sodium hydroxide (30 ml) and methylene chloride (30 ml). Theorganic phase was dried and the solvent removed in vacuo. The residuewas flash chromatographed on silica gel eluting with 40% ethylacetate/60% hexane. Fractions containing the title compound as a freebase were combined and the solvent replaced by diethyl ether. Theetheral solution was treated with gaseous HCl. The volatiles wereremoved in vacuo, and the residue recrystallized from isopropanol toyield 400 mg of the title compound as a white crystalline solid, m.p.134-136° C., [α]_(D)+31.40 (ethanol). NMR δ (CDCl₃): 2.55 (2H, m); 2.7(1H, br.s); 2.8 (3H, s); 3.0 (1H, m); 3.4 (1H, m); 3.9 (2H, br.s); 5.05(1H, m); 7.7 (4H, m) ppm.

EXAMPLE 8 S-(−)-N-Methyl-N-propargyl-1-aminoindan hydrochloride

The title compound was prepared as in Example 7 above, except thatS-(−)-N-propargyl-1-aminoindan (free base) from Example 5 was used asthe starting material. All of the physical and spectral properties ofthe title compound were identical to those in Example 7 except for the[α]_(D)−34.9° C. (ethanol).

EXAMPLE 9

Tablet Composition N-Propargyl-1(R)-aminoindan Hydrochloride  7.81 mg*Pregelatinized starch NF 47.0 mg Lactose NF hydrous 66.0 mgMicrocrystalline cellulose NF 20.0 mg Sodium starch glycolate NF 2.99 mgTalc USP  1.5 mg Magnesium stearate NF  0.7 mg*Equivalent to 5.0 mg of N-propargyl aminoindan base.

EXAMPLE 10

Tablet Composition N-Propargyl-1(R)-aminoindan Hydrochloride  1.56 mg*Lactose hydrous 50.0 mg Pregelatinized starch 36.0 mg Microcrystallinecellulose 14.0 mg Sodium starch glycolate 2.14 mg Talc USP  1.0 mgMagnesium stearate NF  0.5 mg*Equivalent to 1.0 mg of N-propargyl aminoindan base.

EXAMPLE 11

Capsule Composition N-Propargyl-1(R)-aminoindan Hydrochloride  5.0 mgPregelatinized starch 10.0 mg Starch 44.0 mg Microcrystalline cellulose25.0 mg Ethylcellulose  1.0 mg Talc  1.5 mgPurified water added as required for granulation.

EXAMPLE 12

Injection Composition N-Propargyl-1(R)-aminoindan Hydrochloride  5.0 mgDextrose anhydrous 44.0 mgHCl added to pH 5Purified water added as required for 1 ml

EXAMPLE 13

Injection Composition N-Propargyl-1(R)-aminoindan Hydrochloride 1.0 mgSodium chloride 8.9 mgHCl added to pH 5Purified water added as required for 1 ml

EXAMPLE 14

Injection Composition N-Propargyl-1(R)-aminoindan Hydrochloride 2.0 mgSodium chloride 8.9 mgHCl added to pH 5Purified water added as required for 1 ml

EXAMPLE 15

Syrup Composition N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mgSucrose 2250.0 mg   Saccarin sodium 5.0 mg Methylparaben 6.0 mgPropylparaben 1.0 mg Flavor 20.0 mg  Glycerin USP 500 mg  Alcohol 95%USP 200 mg Purified water as required to 5.0 ml

EXAMPLE 16

Sublingual Tablets N-Propargyl-1(R)-aminoindan Hydrochloride 2.5 mgMicrocrystalline cellulose 20.0 mg  Lactose hydrous 5.0 mgPregelatinized starch 3.0 mg Povidone 0.3 mg Coloring agent q.s. Flavorq.s. Sweetener q.s. Talc 0.3 mg

Blend the excipients and the active and granulate with an ethanolsolution of Providone. After drying and weighing, it is blended with thetalc and compressed.

EXAMPLE 17

PAI Sublingual Tablets N-Propargyl-1(R)-aminoindan Hydrochloride 5.0 mgMicrocrystalline cellulose 15.0 mg  Pregelatinized starch 12.0 mg  Ethylcellulose 0.3 mg Talc 0.3 mgPurified water added as required for granulation.

EXAMPLE 19

Tablet Composition N-Propargyl-1(R)-aminoindan Hydrochloride  5.0 mgLevodopa 100.0 mg  Carbidopa 25.0 mg Pregelatinized starch 24.0 mgStarch 40.0 mg Microcrystalline cellulose 49.5 mg Col. D & C Yellow No.10  0.5 mg Col. D & C Yellow No. 6 0.02 mgAlcohol USP added as required for granulation.

EXAMPLE 19

Tablet Composition N-Propargyl-1(R)-aminoindan Mesylate  7.81 mg*Pregelatinized starch NF 47.0 mg Lactose NF hydrous 66.0 mgMicrocrystalline cellulose NF 20.0 mg Sodium starch glycolate NF 2.99 mgTalc USP  1.5 mg Magnesium stearate NF  0.7 mg*Equivalent to 5.0 mg of N-propargyl aminoindan base.

EXAMPLE 20

Tablet Composition N-Propargyl-1(R)-aminoindan Mesylate  1.56 mg*Lactose hydrous 50.0 mg Pregelatinized starch 36.0 mg Microcrystallinecellulose 14.0 mg Sodium starch glycolate 2.14 mg Talc USP  1.0 mgMagnesium stearate NF  0.5 mg*Equivalent to 1.0 mg of N-propargyl aminoindan base.

EXAMPLE 21

Capsule Composition N-Propargyl-1(R)-aminoindan Mesylate  5.0 mgPregelatinized starch 10.0 mg Starch 44.0 mg Microcrystalline cellulose25.0 mg Ethylcellulose  1.0 mg Talc  1.5 mgPurified water added as required for granulation.

The following Examples and the accompanying Tables and Figures relate tobiological experiments carried out in accordance with this invention.

EXAMPLE 22 Inhibition of MAO Activity in vitro

Experimental protocol

The MAO enzyme source was a homogenate of rat brain in 0.3M sucrose,which was centrifuged at 600 g for 15 minutes. The supernatant wasdiluted appropriately in 0.05M phosphate buffer, and pre-incubated withserial dilutions of compounds: R(+)-PAI, S(−)-PAI and racemic PA: for 20minutes at 37° C. ¹⁴C-Labelled substrates (2-phenylethylamine,hereinafter PEA; 5-hydroxytryptamine, hereinafter 5-HT) were then added,and the incubation continued for a further 20 minutes (PEA), or 30-45minutes (5-HT). Substrate concentrations used were 50 μM (PEA) and 1 mM(5-HT). In the case of PEA, enzyme concentration was chosen so that notmore than 10% of the substrate was metabolized during the course of thereaction. The reaction was then stopped by addition of tranylcypromine(to a final concentration of 1 mM), and the incubate filtered over asmall column of Amberlite CG-50 buffered to pH 6.3. The column waswashed with 1.5 ml water, the eluates pooled and the radioactive contentdetermined by liquid scintillation spectrometry. Since the aminesubstrates are totally retained on the column, radioactivity in theeluate indicates the production of neutral and acidic metabolites formedas a result of MAO activity. Activity of MAO in the sample was expressedas a percentage of control activity in the absence of inhibitors aftersubtraction of appropriate blank values. The activity determined usingPEA as substrate is referred to as MAO-B, and that determined using 5-HTas MAO-A.

Results

Inhibitory activity of R(+)-PAI, S(−)-PAI and racemic-PAI were examinedseparately in vitro, and the results of typical experimental runs areshown in FIGS. 1 and 2. The entire experiment was repeated three times.Concentrations of inhibitor producing 50% inhibition of substratemetabolism (IC-50) were calculated from the inhibition curves, and areshown in Table 1B. From this data it can be seen that:

-   -   (a) the R(+)-PAI is twice as active as the racemate for        inhibition of MAO-B;    -   (b) the R(+)-PAI is 29 times more active for inhibition of MAO-B        than MAO-A;

(c) the S(−)-PAI is only ⅙, 800 as active as the R(+)PAI for inhibitionof MAO-B, and shows little or no selectivity between MAO-B and MAO-A.TABLE 1A IC-50 (nM) VALUES FOR INHIBITION OF MAO-A AND MAO-B BYRACEMIC-PAI AND THE R(+) AND S(−) ENANTIOMERS THEREOF IN RAT BRAINHOMOGENATE IN VITRO IC-50 (nM) MAO-A MAO-B S(−)PAI R(+)PAI Rac S(−)PAIR(+)PAI Rac 26000 73 140 17000 2.5 5

The results of the same experiments using R(+) and S(−) MPAI(N-methyl-N-propargyl-1-aminoindan) are reported in Table 1B. Each ofthe enantiomers of MPAI is less selective in MAO-A and MAO-B inhibitionthan R(+) PAI. Furthermore, R(+)-MPAI is only five times as active asS(−)-MPAI in MAO-B inhibition, in contrast to R(+)-PAI which is about7000 times as active as S(−)-PAI in this assay. TABLE 1B IC-50 (nM)VALUES FOR INHIBITION OF MAO-A AND MAO-B BY T

R(+) AND S(−) ENANTIOMERS OF MPAI IN RAT BRAIN HOMOGENAT IN VITRO IC-50(nM) MAO-A MAO-B S(−)MPAI R(+)MPAI S(−)MPAI R(+)MPAI Compound: 70 3 5010

Some experiments were also carried out with human cerebr

cortical tissues obtained 6 hours post-mortem, and treat

as described above. The results of such an experiment a

shown in FIG. 3, where R(+)-PAI, S(−)-PAI, and racemic. P

are as defined herein.

EXAMPLE 23 Inhibition of MAO Activity in vivo: Acute Treatment

Experimental protocol

Rats (male Sprague-Dawley-derived) weighing 250±20 g we

treated with one of the enantiomers or the racemic form

PAI by intraperitoneal injection (ip) or oral gavage (p

and decapitated 1 h or 2 h later respectively. Groups

three rats were used for each dose level of inhibitor, a

MAO activity determined in brain and liver using the gene

technique described above. The amount of protein in ea

incubation was determined using the Folin-Lowry method,

enzyme activity calculated as nmol of substrate metaboliz

per hour of incubation for each mg of protein. Activity

MAO in tissues from animals treated with inhibitors

expressed as a percentage of the enzyme activity in a gr

of control animals administered vehicle (water for o

administration, 0.9% saline for ip injection) and killed as above.

Results

None of the dose levels used with the inhibitor drugs produced anyobvious behavioral alteration. The results are depicted in FIGS. 4 to11. Following i.p. administration, compound R(+)PAI produced 90%inhibition of brain MAO-B activity at a dose of 0.5 mg/kg. The same doseproduced only 20% inhibition of MAO-A activity. By oral administration,the same dose of R(+)PAI produced 80% inhibition of MAO-B with nodetectable inhibition of MAO-A. Essentially similar results were seenfor inhibition of hepatic MAO, as for brain MAO. The doses producing 50%inhibition of MAO-A and MAO-B (IC-50) were calculated from theinhibition curves, and are shown in Table 2. These data show: (a) thatMAO inhibitory activity of R(+)PAI is maintained in vivo in the rat; (b)that selectivity for inhibition of MAO-B, as opposed to MAO-A, byR(+)PAI is maintained in vivo; (c) that the much greater activity of the(+)-enantiomer as opposed to the (−)-enantiomer, is maintained in vivo;(d) that the compounds are effectively absorbed after oraladministration; and (e) that the compounds effectively pass theblood-brain barrier, and effectively inhibit brain MAO. The fact thatR(+)-PAI was about twice as active as the racemic compound forinhibition of MAO-B is a reflection of the extremely low activity ofS(−)-PAI for inhibition of MAO-B. TABLE 2 IC-50 VALUES (mg/kg) FORINHIBITION OF MAO-A AND MAO-B BY R(+)-PAI, S(−)-PAI OR RACEMIC-PAI, INTHE RAT FOLLOWING INTRAPERITONEAL (I.P.) INJECTION OR ORALADMINISTRATION (P.O.) IC-50 (mg/kg) MAO-A MAO-B Compound: S(−)PAIR(+)PAI Rac S(−)PAI R(+)PAI Rac I.P. BRAIN >10 1.2 2.5 >10 0.07 0.22I.P. LIVER >10 5 5 >10 0.06 0.11 P.O. BRAIN >10 >5 >5 >10 0.17 0.29 P.O.LIVER >10 >5 >5 >10 0.05 0.09(Rac = Racemic PAI)

EXAMPLE 24 Inhibition of MAO Activity in vivo: Chronic Treatment

Experimental protocol

Rats (specifications as in Example 23, 4 animals for each dose level)were treated with R(+)PAI or the racemic mixture at three dose levels(0.05, 0.1 and 0.5 mg/kg) by oral administration, one dose daily for 21days, and decapitated 2 hours after the last dose. The activities of MAOtypes A and B were determined in brain and liver as described in Example23.

Results

A daily dose of 0.1 mg/kg of compound R(+)PAI produced a good degree ofselective inhibition, with more than 80% inhibition of brain MAO-B and20% or less inhibition of brain MAO-A. At the higher dose of 0.5 mg/kgdaily, MAO-A was still inhibited by less than 50% (FIGS. 12 and 13).Hepatic MAO showed a similar degree of selective inhibition (FIGS. 14and 15). Compound R(+)PAI was again more potent than the racemic mixtureby a factor of about twofold. In the case of brain MAO, R(+)PAI had abetter degree of selectivity for inhibition of MAO-B than did theracemic mixture.

These results show that selectivity of MAO-B inhibition can bemaintained following chronic treatment with the compounds. As with otherirreversible inhibitors, the degree of enzyme inhibition is greater withchronic treatments than that following a single dose of the drug.Compound R(+)PAI shows a better degree of selectivity for inhibition ofbrain MAO-B than the racemic mixture.

EXAMPLE 25 Irreversible Nature of MAO Inhibition

Experimental protocol

A single dose of compound R(+)PAI (1 mg/kg) was administered by i.p.injection to groups of 4 rats, and the animals killed 2, 6, 18, 24, 48and 72 hours later. Activity of MAO-B was determined in whole braintissues as described hereinabove.

Results

The results are shown in FIG. 16. Maximal inhibition of MAO-B wasattained at 6 hours after injection. MAO activity had only returned to30% of control activity at 72 hours after injection. This experimentdemonstrates the irreversible nature of the MAO inhibition by R(+)PAI.

EXAMPLE 26 Potentiation of Tyramine Pressor Effect in Conscious Rats

Experimental protocol

Rats were anesthetized with a mixture of pentobarbital (30 mg/kg) andchloral hydrate (120 mg/kg) by intraperitoneal injection. The leftcarotid artery and jugular vein were cannulated with fine polytenetubing (artery) or fine silicone rubber tubing connected to polyethylenetubing (vein), the distal end of which was brought under the skin to ananchor point behind the neck. The tubing was filled with heparinizedsaline solution, and plugged with a fine steel rod. The animals weretreated with 20 mg chloramphenicol by intramuscular injection andallowed to recover from the operation overnight. The following day, therats were placed in a high-walled container permitting free movement.The arterial catheter was connected to a pressure transducer via a 100cm length of saline-filled, fine-bore polyethylene tubing, and thevenous catheter connected to a 1 ml syringe via a similar length oftubing, which, together with the syringe, contained a solution oftyramine hydrochloride in saline (1 mg/ml). Following an equilibrationperiod of 30 to 40 minutes, tyramine injections (50 or 100 μg) weregiven, and blood pressure responses recorded. An interval of at least 15minutes was maintained between injections after return of blood pressureto control values. Control pressor responses were established, then oneof the drugs was injected intraperitoneally, and tyramine responses wererepeated over the next 4 hours. The area under the blood pressureresponse curve was estimated, and the ratio of this area after treatmentto before treatment and to 1 to 3 hours after injection of thecompounds, was determined using the average of 3 to 4 values obtained inthe control period.

Results

The results are shown in Table 3. Compound R(+)PAI at a dose of 1 mg/kg(which causes complete inhibition of MAO-B in brain and liver, and 40 to50% inhibition of MAO-A in these tissues) caused no significantpotentiation of tyramine pressor response. At the higher R(+)PAI dose of5 mg/kg (which causes more extensive inhibition of MAO-A in brain andperiphery), there was a significant potentiation of the tyramine pressorresponse, which was similar in extent to that produced by the same doseof deprenyl, and less than that produced by clorgyline (at a dose whichinhibits hepatic MAO-A activity by over 85%). TABLE 3 POTENTIATION OFTYRAMINE PRESSOR EFFECT IN CONSCIOUS RATS BY MAO INHIBITORS Ratio AreaUnder Dose No. of rats Pressor Response Inhibitor (mg/kg) (n) Curve;After/Before SEM* Saline 12 1.25 0.28 Clorgyline 2 6 10.39 2.13(−)Deprenyl 1 2 1.15 (+)Deprenyl 5 3 2.36 0.16 R(+)PAI 1 3 1.38 0.7R(+)PAI 5 3 3.49 0.98*SEM = standard error of the mean

From this experiment it can be concluded that compound R(+)PAI causes nopotentiation of the tyramine pressor effect at a dose which effectivelyinhibits MAO-B.

EXAMPLE 27 Suppression of MPTP-Induced Dopaminergic Toxicity by R(+)PAI

1-Methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin thatdamages nigrostriatal dopaminergic neurons in several mammalian species,including mice, and produces a Parkinsonian syndrome in humans andprimates. A crucial initial step in the mechanism of its neurotoxicityinvolves conversion of MPTP to its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+). This reaction is catalyzed by the enzyme MAO-Band probably takes place outside of dopaminergic neurons, mainly inglia. It is known that MPTP is both a substrate and an irreversibleinhibitor of MAO-B. Pretreatment of experimental animals with MAO-Binhibitors such as deprenyl or pargyline protects against and preventsthe MPTP-induced damage to nigrostriatal neurons because the oxidativeconversion of MPTP to MPP+ is blocked. The progressive nigrostriataldegeneration in Parkinson's may be due to exposure toenvironmentally-derived exogenous MPTP-like neurotoxins. In such cases,there is an additional strong indication of initiation of sustainedtreatment with an MAO-B inhibitor from the very early stages ofParkinson's disease in the hope that it will neutralize the damagingeffects of such yet putative MPTP-toxins, and thus arrest or slow downthe progression of the illness. A successful MAO-B inhibitor drug iscurrently judged by its ability to block MPTP-induced damage tonigrostriatal dopaminergic neurons in vivo. The (−) and (+) enantiomersof PAI were therefore tested for their potency in preventing orattenuating the MPTP-induced striatal dopamine depletions in mice.

Experimental Protocol

Male C57 black mice (20-25 g weight) were (a) injected with MPTP.HCl (30mg/kg dissolved in distilled water, s.c.), or vehicle alone, or one hourafter pretreatment with the (−) or (+) isomers of PAI (2.5 mg/kg, i.p.),or with deprenyl (5 mg/kg, i.p.), and (b) decapitated 5 days later.Brains were removed and corpora striata dissected on an ice-cold glassplate and frozen on dry ice. Striatal tissues were homogenized in 0.1 Mperchloric acid, and deproteinized aliquots containingdihydroxybenzylamine as an internal standard were assayed for dopamineand its major metabolite 3,4-dihydroxy-phenylacetic acid (DOPAC) usingHPLC with electrochemical detection.

Results

Table 4 shows the results of this experiment. Treatment with MPTP aloneproduced marked striatal dopamine (DA) and DOPAC depletions. Treatmentwith the (−) and (+) enantiomers of PAI or with (−) deprenyl did notaffect striatal DA concentrations. Pretreatment with the (−) isomer ofPAI did not affect the MPTP-induced DA and DOPAC levels in the striatum.The (+)-isomer of PAI given before MPTP completely abolished thereduction in striatal DA and DOPAC levels produced by the toxin. At adose of 2.5 mg/kg, (+)PAI was equipotent to (−) deprenyl (5 mg/kg) inits protective effect. TABLE 4 EFFECT OF PRETREATMENT WITH THE (−) AND(+) ENANTIOMERS OF THE MAO-B INHIBITOR PAI ON THE STRIATAL DA AND DOPACDEPLETIONS INDUCED BY MPTP IN MICE IN VIVO DA DOPAC (ng/mg protein)Control 162.8 ± 7.2 8.4 ± 0.5 MPTP  53.1 ± 6.2 3.2 ± 0.3 (−)PAI 174.0 ±4.8 7.5 ± 0.2 (−)PAI + MPTP  53.4 ± 6.9 7.0 ± 0.6 (+)PAI 185.0 ± 6.9 3.3± 0.3 (+)PAI + MPTP  177.8 ± 14.4 6.0 ± 0.3 (−)Deprenyl 170.6 ± 7.1 5.6± 0.3 (−)Deprenyl + MPTP 197.0 ± 8.0 6.4 ± 0.5Above values for DA and DOPAC expressed as Mean ± S.E.M. and number ofrats. n = 7-11 in each group.

These results indicate that the R(+)PAI is an excellent MAO-B inhibitorin vivo, and is of especially great potential for the treatment ofParkinson's disease.

While the invention has been described with reference to theaforementioned Examples and the accompanying Tables and Figures, it isnot restricted thereto. Various modifications and applications of theinvention are possible. For example, (R)-PAI may be combined, in asynergistic way, with α-tocopherol (a vitamin E derivative) for thetreatment of Parkinson's disease.

EXAMPLE 29 Effect of PAT Enantiomers on Amphetamine Induced StereotypeBehavior in Senescent Rats

Amphetamine is known to induce stereotypic behavior (Sulser, F., andSanders-Bush, E,, Ann. Rev. Pharmacol., 11, 209-230 (1971)) by themobilization of endogenous dopamine. Amphetamine is not metabolized byMAO-B. Inhibition of MAO-B by an effective inhibitor and administrationof amphetamine cause release of dopamine which will not undergodegradation by the inhibited MAO-B. Thus, an increase of synapticdopamine is expected after administration of amphetamine and effectiveMAO-B inhibitor leading to an increase in stereotypebehavior-potentiation of the amphetamine effect. The extent of thisbehavior is rated in accordance with the number of lateral headmovements over a period of 1 minute.

Experimental Protocol

The test compound was administered at a dose of 0.5 mg/kg/day indrinking water, 24 hours before the infliction of hypoxia (92%nitrogen+8% oxygen for 6 hours). Following that, amphetamine wasinjected s.c. at a dose of 0.5 mg/kg. 45 minutes later, lateral headmovements were counted.

Results

The results of these experiments are shown in Table 5. TABLE 5 EFFECT OFPAI ISOMERS ON AMPHETAMINE-INDUCED STEREOTYPE BEHAVIOR IN SENESCENT RATS(CONTROL AND HYPOXIA LESIONED) Stereotype Group Treatment BehaviorRating Control (6) — 87 ± 10 Control (5) (+)PAI 126 ± 16* Control (4)(−)PAI 94 ± 18 Hypoxia lesioned (5) — 93 ± 12 Hypoxia lesioned (6)(+)PAI 143 ± 6* Numbers in parentheses are numbers of animals tested.*P < 0.001 with respect to untreated hypoxia group or untreated controlgroup correspondingly.

The results in Table 5 indicate that (+)PAI caused significantpotentiation of the amphetamine-induced stereotype behavior in bothhypoxia-lesioned and control rats. (−)PAI was totally inactive in thisrespect. These behavioral in vivo results corroborate previousbiochemical findings that (+)PAI is an active inhibitor of MAO-B in thebrain while (−)PAI is inactive in this respect.

EXAMPLE 29

Effect on R(+)-PAT on the Improvement or Restoration of Memory

Newborn rat pups subjected to a brief episode of anoxia and then allowedto resume their growth in a normal way develop a long-lasting impairmentof memory (Speiser, et al., Behav. Brain Res., 30, 89-94 (1988)). Thismemory impairment is expressed as an inferior performance in the passiveavoidance test.

The effect of R(+)-PAI and S(−)-PAI on the improvement or restoration ofmemory was investigated in the passive avoidance test. If the drug iseffective, it increases the latency of response to enter a darkcompartment or chamber where an electroshock has been experiencedearlier by the rat being tested. The latency of the maximal response is300 seconds.

Experimental Protocol

Young rats were subjected to post-natal anoxia as described in Example27. R(+)-PAI or S(−)-PAI were administered according to one of thefollowing protocols.

Protocol A—Nursing mothers were given a dose of either isomer of 1-1.5mg/kg/day in drinking water until weaning at 21 days. Following that,the weaned offsprings were directly treated with the same dose for 20days. Treatment was terminated at 40 days and the test was performed at60 days, that is 20 days after the last dose of the drug.

Protocol B—The dose was reduced to 0.5 mg/kg/day administered to thenursing mother until weaning at 21 days, then directly to the young ratsto 60 days at which time the test was performed.

Passive Avoidance Test—The apparatus consisted of a lit chamberadjoining a dark chamber and a sliding door separating the two. Attraining, a rat was placed in the lit chamber for 30 seconds, and thenthe door was opened. The rat moved to the dark chamber with a latencythat was recorded. Upon entry of the rat into the dark compartment, thedoor was closed and a 0.3 mA foot-shock was delivered for 3 seconds.

Retention (memory) after 48 hours was determined by repeating the testand recording the latency to step through from light to darkness to anarbitrary maximum of 300 seconds.

Results

The results of these experiments are shown in Table 6. TABLE 6 EFFECT OFPAI ISOMERS ON PASSIVE AVOIDANCE RESPONSE IN YOUNG RATS (60-DAYS OLD)Before After Group Treatment Electroshock Electroshock PROTOCOL AControl − 49 ± 13 201 ± 111 Control (+)PAI 49 ± 19 220 ± 100(+9%)*Control (−)PAI 48 ± 13 192 ± 116 Anoxia-lesioned − 45 ± 11 183 ± 109Anoxia-lesioned (+)PAI 49 ± 10 239 ± 99(19%)* Anoxia-lesioned (−)PAI 55± 27 179 ± 123 PROTOCOL B Control − 53 ± 20 104 ± 101 Control (+)PAI 48± 11 128 ± 119(+23%)* Anoxia-lesioned − 45 ± 8  119 ± 105Anoxia-lesioned (+)PAI 52 ± 12 137 ± 126(+15%)* Anoxia-lesioned (−)PAI48 ± 19 112 ± 112Figures represent the latency in seconds for entering a dark compartmentwhere an electroshock had been first experienced by the rat tested.*The indicated percent increases are with respect to the correspondinganoxia or control groups.

The experimental results indicated that (+)PAI but not (−) PAI iseffective in improving the memory of anoxia-lesioned and control rats.Drugs active in this test are considered to be potentially useful fortreatment of various memory impairment disorders, dementia andespecially senile dementia of the Alzheimer's type.

EXAMPLE 30 Effect of R(+)-PAI on the Anoxia-Induced Hyperactive Syndromein Juvenile Rats

Rats that had been exposed postnatally to anoxia and then left to growunder normal conditions show increased motor activity in the open fieldat the age of 10-42 days (Hertshkowitz, et al., Dev. Brain Res., 7,145-155 (1983)).

The effect of R(+)PAI and S(−)PAI on such hyperactive syndrome wasinvestigated.

Experimental Protocol

Anoxia was performed on rat pups on the first post-natal day. They wereplaced in a glass chamber and exposed to 100% nitrogen for 25 minutes.They were resuscitated by intermittent massage softly applied to thechest and then returned to their respective mothers. Control ratsreceived the same treatment but with air instead of nitrogen.

The R(+)-PAI or S(−)-PAI (0.5 mg/kg/day) was administered to the nursingmothers in drinking water, thereby transferred to the sucklings throughmilk.

Locomotion was measured in 6 fully computerized cages (28×25 cm) byrecording the number of crossings over a given period of time. Crossingsof grid infrared beams at 4-cm intervals initiated electrical impulseswhich fed a counter. Recordings of motor activity were made at the agesof 15 and 20 days, over a period of 15 minutes.

Results

The experimental results are given in Table 7. TABLE 7 EFFECT OF EACH OFTHE TWO ENANTIOMERS ON THE ANOXIA-INDUCED HYPERACTIVE SYNDROME 15-dayold 20-day old Group Treatment rats rats Control − 414 ± 192(11) 808 ±212(12) Control (+)PAI 254 ± 149(11)c 719 ± 110(13) Anoxia- − 482 ±119(7) 858 ± 96(9) lesioned Anoxia- (+)PAI 276 ± 186(15)a 737 ± 150(16)clesioned Anoxia- (−)PAI 334 ± 196(5) 778 ± 232 (6) lesioned− Numbers in parenthesis are numbers of animals tested. The figures arethe numbers of crossings of infrared beam grid in the activity cage overa period of 15 minutes.^(a)P < 0.001 compared to anoxia untreated group.^(b)P < 0.05 compared to anoxia untreated group.^(c)P < 0.05 compared to control group.

These results indicate that chronic oral treatment with R(+)−PAI at adose of 0.5 mg/kg administered to the nursing mother and reaching themilk-fed offspring significantly improved the hyperactive syndrome.Consequently, R(+)−PAI is a potentially useful drug for the treatment ofthe hyperactive syndrome in children.

EXAMPLE 31 Stability Differences Among Ten Salts of PAI

Stability is an important factor in the selection of an optimal salt asa therapeutic drug. Different salts may alter the physicochemical andbiological characteristics of a drug and can have a dramatic influenceon its overall properties. (Berge, S. M., et al., J. Pharm. Sci. 66, 1(1977); Gould, P. L., Int. J. Pharmaceutics, 33, 201 (1986)).

Experimental

Synthesis of PAT salts

A solution of an appropriate acid (1 mol-eg.) in 2-propanol was added toa solution of PAI (1 mol-eq.) while stirring in 2-propanol (Ar, BHT).The salt formed was filtered, washed with 2-propanol and ether, anddried under low pressure. Yields were between 70 to 90%. An exception inpreparing PAI acetate involved using ether as the solvent.

Analytical methods

The chromatographic separations were carried out using a Lichrosphere 60RP select B 5μ 125×4 mm (Merck) column, an HPLC (Jasco BIP-1) equippedwith a L-4200 UV-Vis detector (Merck-Hitachi) set to 210 nm, and aD-2500 chromato-integrator (Merck-Hitachi). The eluent and diluentconsisted of 80% distilled water/20% acetonitrile (HPLC grade), and 0.07M perchloric acid adjusted to pH 2.5 with aqueous ammonia. The flow rateused was 1 ml/min, the appropriate PA: salt solution concentration was250 μg/ml, and 20 μl of the solution were injected onto thechromatographic system.

The melting range was measured with an automatic apparatus (Mettler FP80) and thermo-gravimetric analysis was performed on a Mettler TA 3000system at a rate of 10° C./min in the applicable range. Solubility wasdetermined by an appropriate dilution of the supernatant from asaturated PAI salt water solution and measured in a UVIKON 941 (Kontron)UV-Vis spectrophotometer. The salt form (mono- or di-salt) was obtainedby elemental analysis using standard equipment for C, H, N and Sdetermination. The pH was measured in a it aqueous solution of the PAIsalts.

Results

The characterization of the various salts are summarized in Table 8.TABLE 8 PHYSICOCHEMICAL PROPERTIES OF PAI SALTS Melting Solubility rangeSalt PAI-salt pH mg/ml (° C.) % Wt. loss form m.w. tartarate 5.5  33176.2-177.3 LT 0.1 di 492 mesylate 4.3 635 156.8-157.6 0.1 mono 267maleate 4.0 NLT 1000 87.2-87.8 0.1 mono 287 sulphate 3.9 485 159.4-161.13.2 di 440 chloride 4.2 238 177.0-180.0 LT 0.5 mono 207 tosylate 4.460-70 129.3-129.9 LT 0.1 mono 343 fumarate 3.5  95 125.4-126.2 0.2 mono287 phosphate 7.0 NLT 720 109.5-110.4 n.a. n.a. n.a. esylate 2.4 NLT 300n.a. n.a. mono 279 acetate 6.1 NLT 720 69.2-69.7 0.4 mono 231n.a. = not available

Comparative stability studies were carried out under sets of severalaccelerating conditions: I) heating at 80° C. for 72, 96 or 144 hours;and II) reflux in isopropanol or 30 hours. The degradation productsdeveloped were measured by HPLC and confirmed by TLC. The results arepresented in Table 9 with the relative retention time (relative to thePAI peak; RRT) as an area percentage relative to to

integrated peak area. TABLE 9 DEGRADATION PRODUCTS DEVELOPED IN PAISALTS UNDER SHOR TERM CONDITIONS Reflux in 80 C./72 h 80 C./144 h iPrOH/Salt RRT^(a) %^(b) RRT % RRT % sulfate ND^(c) ND ND ND 0.47 0.22 0.600.72 phosphate 0.60 0.22 0.60 0.57 0.60 2.62 0.74 0.21 1.84 0.20 1.980.73 chloride ND ND ND ND 2.23 0.71 mesylate ND ND ND ND 0.60 0.08maleate 0.60 0.41 n.a. 0.60 2.17 1.27 0.50 0.65 1.35 1.48 0.33 1.29 0.591.81 0.10 1.42 1.30 3.07 1.44 1.50 0.16 4.16 0.10 1.83 0.18 4.84 7.761.98 0.23 4.09 0.65 acetate 0.44 0.10 n.a. 0.60 6.74 0.60 2.56 0.74 0.350.73 0.13 1.76 0.33 1.29 0.71 1.84 0.16 1.55 1.06 1.99 4.17 1.75 21.853.60 0.27 1.96 3.33 2.15 0.08 2.32 0.15 2.83 0.15 3.54 1.82 esylate^(d)ND ND 0.85 0.26 ND ND 1.96 0.31limit of quantitation = 0.08%n.a. = not available^(a)Relative retention time (relative to the PAI peak).^(b)Area percentage relative to total integrated peak area.^(c)No impurities detected.^(d)Ethyl sulfate salt.

The salts were submitted to visual inspection of color an

form. The findings are shown in Table 10. TABLE 10 APPEARANCE OF PAISALTS UNDER DESTRUCTIVE CONDITIONS reflux in Salt 80° C./72 h 80° C./96h 80° C./144 h iPrOH/30 h sulfate off white n.a. off white brown powderpowder powder phosphate brownish n.a. brown brown powder powder powderchloride white n.a. white off white powder powder powder mesylate whiten.a. white white powder powder powder maleate brown brown n.a. brownmelted melted esylate brownish n.a. dark brown dark brown melted meltedmeltedn.a. = not available

These studies show that sulphate, esylate and mesylat

possess significant advantages relative to the other salt

due to good solubility and chemical stability. Of thes

three salts, mesylate is preferable due to its excellen

stability even under destructive conditions.

EXAMPLE 32 Reversal of Haloperidol-Induced Catalepsy in Mice

Male, ICR mice 25-30 g each, were pretreated with either of thefollowing drugs: Saline, (R)-PAI mesylate, or racemic-PAI mesylate. Alldrugs were administered i.p. in a volume of 0.2 mL. Two hours later,haloperidol was injected s.c. at a dose of 6 mg/kg in a volume of0.1-0.2 mL. Motor coordination tests were made at 3 hours after givinghaloperidol, that is, 5 hours after administering the presumedprotective drugs.

Motor coordination tests and rigidity were quantified according to threedifferent parameters: (a) ability to walk the length of a horizontalrod, 80 cm-long; (b) ability to climb down, face down, a vertical rod,80 cm-long; (c) duration of immobility in an unnatural sitting posturewhereby the abdomen of the mouse is pressed against a “wall.” Fullperformance as in haloperidol-untreated mice is given the score of 4 ineach test, or a total of 12 in all tests. Poor performance is given ascore from 1 to 3. A key to score ratings is given in Table 9A. Theeffects of the various agents in antagonizing haloperidol-inducedcatalepsy are given in Table 11. At three hours after haloperidol,(R)-PAI mesylate conferred protection against haloderidol at 5-15 mg/kg,reaching a peak after effect at 7.5-10 mg/kg (activity score=94% ofsaline control) Racemic PAI mesylate conferred partial protection in therange of 7.5-15 mg/kg, and was not active at 5 mg/kg. From FIG. 17, itcan been seen that the dose-effect profile of either (R)-PAI mesylate orracemic PAI is such that an increase in dose beyond 10 mg/kg entails adecrease in effect, but that the racemic mixture is less potentthroughout. This means that racemic PAI mesylate at twice the dose of(R)-PAI mesylate will always be less active than the (R) enantiomer.

Reversal of α-MpT-induced hypokinesia in rats

The drug α-MpT is assumed to inhibit the formation of L-DOPA fromtyrosine, and consequently the formation of dopamine itself. Lack of CNSdopamine is expressed as hypoactivity. Six month-old male Wistar rats(from Harlan Orkack, UK) were pretreated with saline, (R)-PAI Mesylateor Rac PAI Mesylate, at the indicated doses. Two hours later theyreceived i.p. α-MpT at a dose of 100 mg/kg in 0.3-0.5 mL. Controlsreceived saline. Following this, motor activity was recorded in acomputerized activity cage for the duration of 10 hours. The results aregiven in Table 12 and FIG. 18. At 2 mg/kg, (R)-PAI Mesylate restored thelevel of activity to about 90% of the saline-treated rats, but Rac PAIMesylate was not active. In either case, the profile of the dose-effectcurve was bell-shaped, suggesting a decrease in effect with an increasein dose beyond a peak of 2-5 mg/kg. At 5 mg/kg Rac PAI Mesylate couldnot elicit a level of activity comparable to that of (R)-PAI Mesylate at2 mg/kg.

From these measurements, (R)-PAI Mesylate and Rac PAI Mesylate do notshare a similar pattern of activity in the restoration of normokinesiain haloperidol-treated mice and α-Mpt-treated rats. At all dosesstudied, (R)-PAI Mesylate is always more potent that Rac PAI Mesylate atthe corresponding dose. Also, peak activity of Rac PAI Mesylate isalways lower than peak activity of (R)-PAI Mesylate. Thus, Rac PAIMesylate at a given dose is always less effective than (R)-PAI Mesylateat half the same dose. Doubling the dose of Rac PAI Mesylate withrespect to (R)-PAI Mesylate does not produce an effect equivalent tothat of (R)-PAI Mesylate.

Pharmacologically, Rac PAI Mesylate cannot be considered as consistingof 50% active ingredient which is (R)-PAI Mesylate and 50% inertmaterial as diluent. The presence of (S)-PAI in Rac PAI Mesylate has anadverse effect on the activity of (R)-PAI, resulting in a more thantwo-fold decrease in potency. The decrease may be due to a directadverse effect of (S)-PAI on behavioral parameters. TABLE 11 REVERSAL OFHALOPERIDOL-INDUCED CATALEPSY IN MICE WITH (R)-PAI MESYLATE AND RACEMICMESYLATE Mice received each of the test drugs i.p. at the indicateddoses. Two hours later they received haloperidol as described in thetext. The doses shown are for the free base. (R)-PAI Mesylate Rac PAIMesylate Dose, % of % of mg/kg Score + SE n control Score + SE n control1.8  7.2 ± 1 6 60 7.0 ± 0.6 6 59 3.0  6.4 ± 0.5 6 60 5.9 ± 0.7 6 49 5.0 8.7 ± 0.9* 6 73 6.4 ± 0.4 6 53 7.5 11.0 ± 0.4*** 5 92 9.4 ± 0.8++ 6 7810 11.3 ± 0.3*** 6 94 9.2 ± 0.6*** 6 77 15 10.8 ± 0.5*** 5 90 8.8 ± 0.8*6 73 Control   12 ± 0 12 100 saline Haloperidol  6.6 ± 0.3 16 59 aloneStatistical significance with respect to haloperidol alone:*p ≦ 0.05;**p ≦ 0.01;***p ≦ 0.001 by the Student's “t” test.The scores for (R)-PAI are significantly different from those of racemicPAI at 5 mg/kg, p ≦ 0.05; at 10 mg/kg, p ≦ 0.01; and at 15 mg/kg, p ≦0.05.

TABLE 11A KEY TO SCORE RATING OF HALOPERIDOL-INDUCED CATALEPSY IN MICEAND ITS REVERSAL BY VARIOUS AGENTS Vertical Rod: Unable to grasp rodwith limbs 1 Able to grasp but slips down 2 Able to grasp, partly slips,partly climbs down 3 Able to grasp, climbs down using all limbs 4Horizontal Rod: Unable to grasp, falls off rod 1 Able to grasp, unableto walk on rod more than 2 paces 2 Able to grasp, walks half-length ofrod 3 Able to grasp, walks full length of rod 4 Immobility SittingAgainst Wall: Immobility >5 min 1 Immobility 3-5 min 2 Immobility 1-3min 3 Immobility 0.1 min 4Fractional scores are assigned, such as 2.5, when behavior falls betweentwo categories, as between 2 and 3.

TABLE 12 RESTORATION OF MOTOR ACTIVITY IN RATS TREATED WITHα-METHYL-p-TYROSINE (α-MpT) AT 100 mg/kg i.p. Rats received the testdrugs i.p. at the indicated doses. After two hours they received α-MpTand were immediately placed in activity cages. Total motor activity wasautomatically recorded for 10 hours, as described in the text. (R)-PAIMesylate Rac PAI Mesylate Dose, % of % of mg/kg Score + SE n controlScore + SE n control 2 14,132** ± 1457   7 89 9,035 ± 829 6 57 5 12,893*± 1,869  7 81 10,926* ± 8 69 820 7.5 6,679 ± 414  4 42 9,698 ± 557 4 61Control 15,862 ± 1,424 5 100 saline α-Mpt 8,108*** ± 810    6 51 aloneStatistical significance by the Student's “t”,*p ≦ 0.01;***p ≦ 0.001 for Test drugs + α-Mpt versus α-MpT alone α-Mpt aloneversus control salineThe scores of (R)-PAI versus racemic PAI are significantly different at2 mg/kg, p ≦ 0.01.

EXAMPLE 33 The Effects of (R)-PAT Mesylate Following Closed Head Injuryin Rats

Methods

1. Induction of trauma

Head trauma was induced in male rats under ether anesthesia by a wellcalibrated weight-drop device that falls over the exposed skull,covering the left cerebral hemisphere, 1-2 mm lateral to the midline, inthe midcoronal plane.

2. Evaluation of motor function

One hour after induction of trauma, the rats were tested by a set ofcriteria which evaluated their neurologic outcome (the criteriadescribed by Shohami, et al., J. Neurotrauma, 10, 113 (1993)). Thesecriteria, referred to as the Neurological Severity Score (NSS), consistof a series of reflexes and motor functions. Points are given based ondeficits in these criteria. At 24 h the rats were re-evaluated.

3. Evaluation of brain edema

The brains were removed after the second evaluation of motor function(24 h). A piece of tissue (−20 mg) was weighed to yield wet weight (WW).After drying in a desiccator oven for 24 h at 90° C., it was reweighedto yield dry weight (DW). Water percentage in the tissue was calculatedas (WW-DW)×100/WW.

4. Drug treatment

(R)-PAI Mesylate was dissolved in water. The rats were injectedintraperitoneally at a dose of 0.1 mg/kg, 0, 4, 8 and 12 h afterinduction of head trauma. Control rats were treated with water at thesame times.

Results

The NSS, which measures the “clinical” status of the rats, was almostidentical in the treated and nontreated groups at 1 hour after the headinjury, but significantly lower at 24 hours in the (R)-PAImesylate-treated rats (Table 13). These results indicate that PAImesylate is effective in improving motor function recovery followingclosed head injury in rats.

At 24 hours after trauma, a major edema was found in the hemisphere(85.4% water in the brain of control rats vs. 78.5% in undamaged braintissue). PAI mesylate was effective in reducing edema as verified by itseffect on the percent of water.

In conclusion, the results reported herein demonstrate that (R)-PAImesylate has neuroprotective properties in a model intended to mimichuman nerve injury and to induce trauma to a closed skull. TABLE 13 NSSΔ NSS % H₂0 1 h 24 h (1 h-24 h) in the brain control 15.6 12.3 4.3 ± 0.585.4 ± 0.4 (n = 6) (R)-PAI 16.7 10.2  6.5 ± 0.7*  82.1 ± 0.6** Mesylate(n = 6)*P < 0.05 (Mann Whitney U-test)**P < 0.005 (t-test)

EXAMPLE 34 Effects of PAT Mesylate on Prevention of NMDA Induced CellDeath of Cerebellum Cell Cultures

Results of in vitro assays

Procedures: Cultures of mechanically dissociated neonatal ratcerebellum, The cerebella are dissected aseptically from 6 or 7-day-oldrat pups and placed in a 15 ml sterile plastic conic tube containing 3ml of enriched medium (the medium is made up of Dulbecco's modifiedEagle's medium (DMEM) with high glucose concentration (1 g/l), 2 mM(v/v) L-glutamine, antibiotic antimitotic mixture, and enriched with 15%(v/v) heat-inactivated fetal calf serum). The cerebella are thendissociated after 20-25 passages through a sterile 13 gauge, 10 cm longstainless steel needle attached to a 5 ml syringe with an inserted 45 μmpore size nylon sieve. The dissociated cells are centrifuged at 200 gfor 5 minutes, the supernatant discarded and the cells resuspended inenriched medium. The cell viability is determined by the trypan blueexclusion test. The cells are then plated at a density of 200/mm² onpoly-L-lysine-coated surfaces (Poly-L-lysine-coated glass coverslips areprepared at least 1 hour before plating, by immersing in a steriledistilled water solution containing 15 μg/ml poly-L-lysine, and justbefore use, washing with sterile water and drying), covered withenriched medium and incubated at 37° C. in an atmosphere of 5% CO₂ inair and 100% humidity. After 4 days in culture, the media are replacedwith media containing the desired test compounds. Experiments are donein duplicate and repeated 2 or 3 times. After determining the testcompound toxic dose-response, four groups are compared: (I) control(enriched medium alone), (II) test compound (one subgroup for eachconcentration (2 concentrations are tested)), (III) N-methyl-D-aspartate(NMDA, exposure to a concentration of 1 mM for 3 h) as the cytotoxicchallenge, (IV) test compound plus NMDA (one subgroup for each of the 2concentrations of test compounds), (V) control group to test the effectof solvent (in which the test compound is dissolved), and (VI) anadditional “positive control” group of spermine (0.01 μM dissolved inculture medium) plus NMDA. Nerve cell survival is evaluated by phasecontrast microscopy and trypan blue staining after 24 h.

Results

It is well established that glutamic acid (Glu) possesses neurotoxicproperties which are expressed in several neurological disordersincluding epilepsy and stroke, and most likely also in brainneurodegenerative diseases such as Parkinson's disease, Alzheimer'sdisease and traumatic brain injury. The neurotoxic effects of Glu aremediated by membrane bound Glu receptors, such as N-methyl-D-asparate(NMDA) receptors.

The results, as shown in Table 14, demonstrate that 10 μM of (R)-PAImesylate increased the survival of cerebellum cells by 27 percentfollowing 1 μM NMDA exposure. These in vitro results support the in vivoeffects of (R)-PAI mesylate presented in Examples 33 and 35, indicatingthat this drug has neuroprotective properties against neurotoxicconcentration of NMDA. TABLE 14 NEUROPROTECTIVE EFFECT OF (R)-PAIMESYLATE ON PREVENTION OF NMDA-INDUCED CELL DEATH OF CEREBELLUM CELLSExperimental Group Surviving Cells Percent Protection CerebellarCultures (Toxicity TD₂₅ = 30 μM; TD₅₀ = 85 μM; TD₁₀₀ = 320 μM) Control100 Solvent 97 NMDA 10 Solvent + NMDA 10 0 Compound + NMDA: 1) 0.01 μM +NMDA 12 2 2) 1.00 μM + NMDA 22 12 3) 10.00 μM + NMDA 37 27 Spermine +NMDA 75 65

Values, expressed as the percent of untreated controls, represent theaverage of 2 experiments run in duplicate for culture experiments, andthe mean±SEM of 4 animals for ischemia. The percent protection value isthe effect of the test compound after subtraction of the solvent effect.

EXAMPLE 35 Effects of (R)-PAI Mesylate After Graded Crush of the RatOptic Nerve

Neuroprotective effects of (R)-PAI Mesylate were determined forapplication immediately after crush injury of the optic nerve in theadult rat. Short-term effects were measured metabolically, and long-termeffects electrophysiologically.

Methods

1. Metabolic measurements

a) General. The method is described by Yoles, et al., InvestigativeOphthalmology & Visual Science, 33, 3586-91 (1992). At short terms,metabolic measurements were monitored in terms of the mitochondrialNADH/NAD ratio, which depends on the activity of the electron transportsystem, and thus indicate levels of energy production. Changes inability of the nerve to produce energy as a consequence of injury weredetermined by comparing NADH levels in response to artificial transientanoxic insult before and after the injury.

b) Surface fluorometry—reflectometry. Monitoring of theintramitochondrial NADH redox state is based on the fact that NADH,unlike the oxidized form NAD⁻, fluoresces when illuminated at 450 nm. Aflexible Y-shaped bundle of optic fibers (light guide) was used totransmit the light to and from the optic nerve. The light emitted fromthe nerve was measured at two wavelengths: 366 nm (reflected light) and450 nm (fluorescent light). Changes in the reflected light werecorrelated with changes in tissue absorption caused by hemodynamiceffects and with movements of the optic nerve secondary to alterationsin arterial blood pressure and nerve volume. The fluorescencemeasurements were found to be adequately corrected for NADH redox statemeasurements by subtraction of the reflected light (366 nm) from thefluorescent light (1:1 ratio) to obtain the corrected fluorescencesignal.

c) Animal preparation. Animal utilization was in accord with the ARVOResolution on the use of animals in research. Male Sprague-Dawley (SPD)rats weighing 300-400 g were anesthetized with sodium pentobarbitone (50mg/kg intraperitoneally). With the animal's head held in place by a headholder, a lateral canthotomy was performed under a binocular operatingmicroscope and the conjuctiva was incised lateral to the cornea. Afterseparation of the retractor bulbi muscles, the optic nerve wasidentified and a length of 3-3.5 mm was exposed near the eyeball byblunt dissection. The dura was left intact and care was taken not toinjure the nerve. A special light-guide holder was implanted around theoptic nerve in such a way that the light guide was located on thesurface of the optic nerve 1 mm distal to the injury site. Animals,while still anesthetized, were allowed to recover for 30 minutes fromthe surgical procedures and were then exposed to anoxic conditions. Ananoxic state was achieved by having the rat breathe in an atmosphere of100% nitrogen for 2 minutes after which time it was returned to air. Inorder to evaluate the metabolic activity of the optic nerve, therelative changes in reflected and fluorescent light intensities inresponse to anoxia were measured before and after crush injury.

d) Experimental protocol for crush injury and metabolic measurements.With the aid of calibrated cross-section forceps, a moderate crushinjury was inflicted on the nerve between the eye and the light guideholder at a pressure corresponding to 120 g for 30 sec. Immediatelyafter injury, animals received intraperitoneal injections of water withand without (R)-PAI Mesylate (2 mg/kg). To assess the activity of theenergy production system, NADH response to 2 minutes of anoxia wasmeasured in all animals prior to injury, 30 minutes after injury, andthereafter at hourly intervals up to 4 hours (see FIG. 19).

2. Electrophysiologically Measurements. This method is described byAssia, et al., Brain Res., 476, 205-212 (1989). Animal preparation andoptic nerve injury were preferred as in the metabolic studies.Immediately after injury, animals received a single injection of waterwith or without (R)-PAI Mesylate (0.5 mg/kg). Fourteen days after injuryand treatment, the optic nerves were excised and measuredelectrophysiologically. Prior to removal of optic nerves forelectrophysiological measurement, the rats were deeply anesthetized with70 mg/kg pentobarbitone. The skin was removed from the skull and theoptic nerves were detached from the eyeballs. Subtotal decapitation wasperformed and the skull was opened with a rongeur. The cerebrum wasdisplaced laterally, exposing the intracranial portion of the opticnerve. Dissection was at the level of the nerve, which was transferredto vials containing fresh salt solution consisting of NaCl (126 mM), KCl(3 mM), NaH₂PO, (1.25 mM), NaHCO₃ (26 mM), MgSO₄ (2 mM), CaCl₂ (2 mM),and D-glucose (10 mM), and aerated with 95% O₂ and 5% CO₂ at roomtemperature. The nerves were kept in this solution, in which electricalactivity remained stable for at least 3-4 hours. After 0.5 hours ofrecovery at room temperature, electrophysiological recordings wereobtained from the nerve distal to the crush lesion. The nerve ends werethen connected to two suction Ag-AgCl electrodes immersed in a bathingsolution at 37° C. A stimulating pulse was applied through the electrodeat the proximal end and the action potential was recorded by the distalelectrode. A Grass SD9 stimulator was used for supramaximal electricalstimulation (0.5 pps). The measured signal was transmitted to a MedelecPA36 preamplifier and then to an electromyograph (Medelec MS7, AA7Tamplifier). The solution, stimulator, and amplifier had a common ground.The maximum amplitude of eight averaged compound action potentials(CAPs) was recorded and photographed with a Polaroid camera. The CAPvalues measured in contralateral uninjured nerves served as a reference.

Results

The results demonstrate that (R)-PAI Mesylate applied immediately afteroptic nerve injury blocked the injury-induced reduction in energyproduction. (R)-PAI Mesylate also has a long-term effect measured byelectrophysiological monitoring.

The CAP (compound action potentials) amplitude is directly correlatedwith the number of conducting fibers in the tested segment of the nerve.

(R)-PAI Mesylate significantly attenuated the injury-induced loss ofactivity in the distal segment of the injured nerve, indicating that(R)-PAI Mesylate is a neuroprotective agent or at least slows downdegeneration. TABLE 15 Electrophysiological Measurements CAP amplitude(μV) Group (Mean ± Std. Error.) Vehicle 441 ± 95  N = 13 (R)-PAI 2104 ±313* Mesylate N = 7

EXAMPLE 36 Comparison of Anticonvulsive Properties of R-PAI end S-PAISalts

Both (R)-PAI and (5)-PAI HCl salts have significant anticonvulsantactivities. In mice (i.p. administration) in the maximal electroshocktest (MES test), (S)-PAI HCl has greater anticonvulsant activity(ED₅₀=57 mg/kg) than (R)-PAI HCl (ED₅₀=79 mg/kg). Analogous results wereobserved in rats (p.o. administration). Four out of four rats wereprotected from seizures in the MES test when 50 mg/kg of (S)-PAI HCl wasadministered, whereas three out of four mice were protected after thesame dose of (R)-PAI HCl. With respect to efficacy for Parkinson'sdisease, the enhanced anticonvulsant activity is a detrimental sideeffect. The same trend occurs with the mesylate salts. (S)-PAI Mesylatehas greater anticonvulsant activity than (R)-PAI Mesylate in the MEStest. At doses of 100 mg/kg, (S)-PAI Mesylate protected three out ofthree mice, whereas only one out of three mice was protected with(R)-PAI Mesylate.

The MES test is a classical model to indicate efficacy for partial andgeneralized seizures in humans. The agents' mechanism of action is viatheir ability to prevent the spread of seizures. Some agents, however,that prevent seizure spread have the side effect of lowering seizurethreshold. These agents therefore have both proconvulsive andanticonvulsive side effects.

Results herein show that (S)-PAI Mesylate has proconvulsive activity. Inthe Timed Intravenous Infusion of Metrazol test, 141 mg/kg of (S)-PAIMesylate reduces the time, and therefore the amount of Metrazol,required to induce the appearance of both the first focal seizure andthe onset of clonus. Other agents that are classically used for partialand generalized seizures, such as phenytoin and carbamazepine, do notshow this effect. (H. J. Kupferberg, Epilepsia, 30, s51-s56 (1989)).Likewise, (S)-PAI Mesylate showed a significantly higher acuteneurotoxicity than (R)-PAI Mesylate. At 300 mg/kg, (R)-PAI Mesylate didnot show any neurotoxicity with mice in the rotorod ataxia test. With(S)-PAI Mesylate, four out of four mice showed neurotoxicity andspasticity.

Methods

TD₅₀ (median toxic dose). This test measures neurological deficits bythe rotorod ataxia test. A mouse is placed on a knurled rod rotating at6 rpm. It is then determined whether a mouse has the ability to maintainits equilibrium and stay on the rod for one minute in each of threetrials.

Timed Intravenous Infusion of Metrazol Test. This test measures theminimal seizure threshold of each animal. Metrazol is infused at 0.185mg/ml into the tail veins of mice. The time is then recorded (sec) fromthe start of infusion until the appearance of the first twitch (firstfocal seizure) and onset of clonus (clonic seizure). Proconvulsantsrequire less Metrazol to produce these symptoms and therefore showendpoints at a shorter period of time.

EXAMPLE 37 Peripheral Effects of (R)-PAI and (S)-PAI on theContractility of Intestinal Smooth Muscle Preparations

Peripheral effects of the hydrochloride salts of the enantiomers of PAIwere determined in isolated rabbit or guinea-pig small intestine. Theseobservations provide useful information on their relative peripheralside effects in humans. The first point of contact of the subject withan orally administered drug is the gastrointestinal tract whereconcentrations of the drug are much higher than after absorption anddistribution. In the case of PAI hydrochloride (MW=208), a 10 mg oraldose contained in a liquid volume of about 100 ml would be equivalent toa concentration of about 0.5 mM. In contrast, the therapeutic plasmaconcentration of (R)-PAI hydrochloride is in the nanomolar range.

The effect of the enantiomers of PAI in the isolated rabbit jejunum andthe guinea-pig ileum were determined so as to find out whether theintake of (S)-PAI together with (R)-PAI (as found in racemic PAI) wouldproduce side effects absent in the administration of pure (R)-PAI.(R)-PAI is the preferred enantiomer for the inhibition of MAO-B in thebrain, in view of its potency and high selectivity towards this form ofthe enzyme. (S)-PAI is much less potent than (R)-PAI an this respect andis also not selective toward MAO-B. In principle, its presence in PAIracemate might be tolerated or overlooked provided (S)-PAI is inert atthe recommended doses of (R)-PAI. The results provided in Tables 16-19show that (S)-PAI is not an inert substance. On the contrary, in theguinea-pig ileum, it is a more potent relaxant than (R)-PAI. Hence itsperipheral effects cannot be discounted as negligible. These data showthat there would be fewer peripheral side effects in the administrationof pure (R)-PAI than in the administration of racemic PAI containing anequivalent dose of (R)-PAI. TABLE 16 TYRAMINE POTENTIATION BY EACH OFTHE TWO ENANTIOMERS OF PAI HCl IN RATE JEJUNUM PREPARATION A stretch ofrabbit jejunum, mounted in an organ bath, displays rhythmic contractionsthat are inhibited by norepinephrine but not by tyramine. If however thejejunum is pretreated with a monoamine oxidase inhibitor such as PAI,then tyramine causes relaxation of the spontaneous contractions. Theextent of relaxation can be correlated with the relative potency of theinhibitor. Percent Drug and concentration (μM) relaxation Tyramine alone40 0 Norepiniphrine 0.002 100 (R)PAI alone 0.2-4.0 0 (S)PAI alone0.2-4.0 0 Tyramine 40 after (R)PAI 0.2 67 2 88 40 85-90 after (S)PAI 0.20 2 35 40 33-50

Results

(S)-PAI is much less potent than (R)-PAI as an inhibitor of brain MAO-B.Therefore, (S)-PAI is not a useful agent for the prevention of braindopamine degradation, but can potentiate the tyramine-evoked release ofnorepinephrine in the small intestine. Its activity in the smallintestine is an undesirable side effect as it is expected to increasethe absorption and action of undegraded tyramine. Thus, (S)-PAI is notan inert substance when used together with (R)-PAI as found in racemicPAI. TABLE 17 ANTAGONISM OF BETHANECHOL-INDUCED CONTRACTIONS OF THEGUINEA PIG ILEUM PREPARATION IN THE PRESENCE OF 400 μM OF EACH OF THETWO ENANTIOMERS OF PAI HCl A stretch of guinea-pig ileum mounted in aphysiological solution in an organ bath contracts dose-dependently whentreated with bethanechol which is an enzymatically stable analog of thenatural gastrointestinal neurotransmitter acetylcholine. Thesecontractions are attenuated in the presence of PAI. The data areexpressed in gram-tension. gram-tension Bathenechol (μM) control (R)PAIcontrol (S)PAI) 0.8 0.5 0.2 0.6 0 2 1.5 0.3 2.0 0 4 2.2 0.7 3.0 0 8 4.01.0 3.8 0.6 20 5.6 2.0 3.8 1.2 40 6.2 2.8 3.8 1.7 80 6.2 3.1 3.8 2.6 2006.2 4.3 3.8 2.6

Results

(S)-PAI is almost inactive as a MAO-B inhibitor with respect to (R)-PAI,and hence is not effective in preventing the degradation of braindopamine. However, it is more effective than R(PAI) in the prevention ofthe bethanechol-induced contraction of the small intestine. Thus (S)-PAIis not an inert substance when used with R(PAI) as found in racemic PAI.TABLE 18 ANTAGONISM OF THE HISTAMINE-INDUCED CONTRACTIONS OF THEGUINEA-PIG ILEUM PREPARATION BY EACH OF THE TWO ENANTIOMERS OF PAI HCl Afixed dose of histamine (40 nM) causes a sustain

contraction of a stretch of guinea-pig ileum mounted

physiological solution in an organ bath. Increment

addition of each of the two enantiomers of PAI HCl causes dose-dependentrelaxation of the muscle. Results a

expressed as percent relaxation with respect to the bas

line before addition of histamine, which is taken as 10

relaxation. PAI Percent relaxation concentration μM (R)PAI (S)PAI 2 0 114 0 15 10 0 30 20 20 30 31 33 40 37 36 100 81 71 200 90 300 92 400 10098 700 100 1000 100

Results

(S)-PAI is inactive with respect to (R)-PAI as a MA

inhibitor in the brain, and hence useless for preventing

degradation of brain dopamine, but is more active than

(R) isomer in causing relaxation of intestinal smo

muscle. Thus, (S)-PAI is not an inert substance when taken together withthe (R)isomer as found in racemic PAI. TABLE 19 ANTAGONISM OF THEBETHANECHOL-INDUCED CONTRACTIONS OF THE GUINEA-PIG ILEUM PREPARATION BYEACH OF THE TWO ENANTIOMERS OF PAI HCl A fixed dose of bethanechol (0.8μM) causes a sustained contraction of a stretch of guinea-pig ileummounted in physiological solution in an organ bath. Incremental additionof each of the two enantiomers of PAI HCl causes a dose-dependentrelaxation of the preparation. Results are expressed as percentrelaxation with respect to the base- line before addition of histamine,which is taken as 100% relaxation. PAI Percent relaxation concentrationμM (R)PAI (S)PAI 20  25 40-50 60 25-50 60-70 100 50-70 100 300 100 100

Results

(S)-PAI is inactive with respect to (R)-PAI as a MAO-B inhibitor in thebrain, and hence useless for the prevention of the degradation of braindopamine, but is more active than the (R) isomer in causing relaxationof intestinal smooth muscle. Thus, (S)-PAI is not an inert substancewhen taken together with the (R) isomer as found in racemic PAI.

EXAMPLE 38 Some Effects of [R](+)PAI Mesylate in Middle Cerebral ArteryOcclusion in the Rat as a Model for Stroke

Methods

1.1. Middle cerebral artery occlusion (MCAO) in the rat. A modificationof the procedure described by Tamura et al (1981) was used. Male Wistarrats (Olac England-Jerusalem) 300-400 g each were anesthetized with asolution of Equitesine administered i.p. at a dose of 3 mL/kg.Equitesine consists of 13.5 mL sodium pentothal solution (60 mg/mL), 3.5g chloral hydrate, 1.75 g MgSO₄, 33 mL propylene glycol. 8.3 mL absolutealcohol made up to 83 mL with distilled water. Surgery was performedwith the use of a high magnification operating microscope, model SMZ-2B,type 102 (Nikon, Japan). In order to expose the left middle cerebralartery, a cut was made in the temporal muscle. The tip of the coronoidprocess of mandible was excised as well and removed with a fine rongeur.Craniectomy was made with a dental drill at the junction between themedian wall and the roof of the inferotemporal fossa. The dura matterwas opened carefully using a 27 gauge needle. The MCA was permanentlyoccluded by microbipolar coagulation at low power setting, beginning 2-3mm medial to the olfactory tract between its cortical branch to therhinal cortex and the laterate striate arteries.

After coagulation, the MCA was severed with microscissors and divided toensure complete occlusion. Following this, the temporalis muscle wassutured and laid over the craniectomy site. The skin was closed with arunning 3-0 silk suture. A sham craniectomy operation was performed on aparallel group of rats, but without cauterization of the MCA. During theentire surgical operation (20-25 min) in either group, body temperaturewas maintained at 37 to 38° C. by means of a body-temperature regulator(Kyoristsu, Japan) consisting of a self-regulating heating pad connectedto a rectal thermistor. At 24 hours post surgery a neurological scorewas taken in order to assess the severity of the injury in thedrug-treated rats with respect to their untreated controls. At 46 hours,the animals were anesthetized with Equitesine and the severity of theinjury was visualized by an MRI procedure. The volume of brain tissueincurring damage following ischemia was determined.

1.2. Drug administration

[R](+)PAI Mesylate was administered as an i.p. injection in 0.3-0.4 mLdistilled water, according to the following schedule:

1 mg/kg immediately after surgery.

0.5 mg/kg 2 hours after surgery

1 mg/kg 20-24 hours after surgery

1.3. MRI scan of ischaemic brain lesion

All experiments were performed using a 4.7T BIOSPEC system (BRUKER) (SeeT. Back, et al., “Diffusion Nuclear Magnetic Resnonance Imaging inExperimental Stroke: Correlation with Cerebral Metabolites,” Stroke(February 1994) 25: 494-500). Forty-eight hours after MCAO or shamoperation, every animal was subjected to a fast multislices T1 weightedimaging (TR/TE). (500/25) for positioning. Then multislices T2-weightedimages (3000/80) were acquired (5 contiguous slices, 3 mm thick).

The size and severity of the infarcted area was estimated using thehyperintensity observed in the T2 weighted MRI at 48 hourspost-occlusion or post sham-operation. The following MRI parameters weredetermined for each group of rats:

c. Ischemic area (in mm²)

d. Area of the ischemic hemisphere (in mm² )

e. Area of the unaffected hemisphere (in mm²)

The use of contiguous slices allows the conversion of area units intovolume units by simply multiplying the area value by the slice thickness

1.4. Neurological score

The neurological score consists of the sum total of a series of ratingsassigned to the performance of specific locomotor activities in a givenrat. The scale runs from 0 (fully normal rats) to 13 (fullyincapacitated rats). Most parameters are rated as either 0 (normal), or1 (incapacitated); others are graded. The following tests were used inthe present study:

General observational tests: hypoactivity; sedation; piloerection

Motor reflex. Rats were lifted by the tail about 15 cm above the floor.Normal rats assume a posture in which they extend both forelimbs towardsthe floor and spread the hind limbs to the sides in a trapeze-likemanner. MCAO when severe causes consistent flexion of the contralaterallimb.

Motor ability. This is seen as the ability to grasp a rod 1 cm indiameter by the contralateral limb for 5-15 sec when the rat is lefthanging on the rod through the arm pit.

Motor coordination. Normal rats are able to walk up and down a beam 5 cmwide placed at a moderate slant. Failure to walk the beam in eitherdirection reveals some motor incoordination, lack of balance and limbweakness.

Gait. Ability to restore normal position to either hind contralaterallimb when intentionally displaced while on a narrow beam.

Balance. Ability to grasp and balance on a narrow beam 2 cm wide.

Locomotor activity. Total movements over a period of 15 min in anautomated activity cage.

Ratings assigned to each of the above parameters are given in Table 20.TABLE 20 Neurological scores assigned to each of 10 parameters ofposture and locomotion Parameter Score a. Activity in the home cagenormal = 0 hypoactive = 1 b. Sedation none = 0 marked = 1 c. Piloeretionnone = 0 marked = 1 d. Extension of contralateral good = 0 forelimbtowards floor when flexed limb = 1 lifted by tail e. Spread ofcontralateral hind good = 0 limb when lifted by tail flexed limb = 1(trapezoid posture) f. Grasp rod with contralateral limb good = 0 poor =1 for 5-15 sec. when suspended by the armpit g. Walk on beam 5-cm widegood = 0 poor = 1 h. Restoration of contralateral good = 0 hind and orfore limb to original poor = 1 (one limb) position when intentionally 2(two limbs) displaced i. Grasping and balance on beam good = 0 poor = 12-cm wide j. Motor activity with respect to ≦25% of control 3 control(for 15 min in an automated 26-50% of control 2 activity cage 51-75% ofcontrol 1 76-100% of control 0

2. Results

2.1. Infarct size

The results of the MRI study are summarized in Table 21 and FIG. 20. Theinfarct size was significantly smaller in [R](+)PAI Mesylate-treatedrats (n=9) than in untreated rats (n=10). In the former, the infarctsize was about 60% of that in the untreated animals. TABLE 21 Ischaemicbrain lesion evaluation by MRI T2-SCAN - 48

following MCA-Occlusion and [R](+)PAI Mesylate treatment Wistar Rats.MCA-O + MCA-O [R](+)PAI Mesylate* Infarct size Infarct size Animal No.(mm³) Animal No. (mm³) 1 252 1 94.4 2 272 2 139 3 314 3 240 4 273 4 1375 201 5 137 6 221 6 174 7 358 7 164 8 265 8 171 9 341 9 215 10  236 MEAN± SD 273.3 ± 50.9 MEAN ± SD 163.5 ± 43.9t = 5.0475f = 17p < 0.001[R](+)PAI Mesylate reduces infarct size by 40% significantly*[R](+)PAI Mesylate administered: Time after MCA-Occlusion:0 - 1.0 mg/kg ip;2 hrs - 0.5 mg/kg ip;24 hrs - 1.0 mg/kg ip.

2.2. Neurological score

The neurological score in five [R](+)PAI Mesylate treated rats and sixuntreated rats were determined by a blinded observer. The results aregiven in Table 22 where they are compared with the infarct size in eachanimal as determined by the MRI test, and also in FIG. 21. It can beseen that those animals with the least neurological scores were thosetreated with [R](+)PAI Mesylate. The neurological score was reduced by54% and the infarct size by 36% in [R](+)PAI Mesylate-treated MCAO ratsas compared to untreated ones. TABLE 22 Neurological score of Ratssubmitted to MCA-Occlusion and [R](+)PAI Mesylate treatment withrelation to their ischaemic infarct size. MCA-O MCA-O + [R](+)PAIMesylate*** Animal Neurological* Infarct size** Animal Neurological*Infarct size** No. Score (mm³) No. Score (mm³) 1 5.0 201 1 1.0 137 2 5.0221 2 2.0 174 3 6.0 358 3 4.0 164 4 6.0 265 4 4.0 171 5 8.25 341 5  2.88215 6 5.75 236 MEAN + SD 6.0 ± 1.19 270 ± 65 MEAN + SD 2.78 ± 1.3 172 ±28 Neurological Score Infarct Size t = 4.25 t = 3.34 f = 9 f = 9 p <0.01 p < 0.01 [R](+)PAI Mesylate reduces the neurological score by 53.7%and infarct size by 36.3%. *Examined 24 hrs after MCA-Occlusion.**Evaluated by MRI T2-SCAN 48 hrs after MCA-Occlusion. ***[R](+)PAIMesylate administered: Time after MCA-Occlusion: 0 1.0 mg/kg ip; 2 hrs0.5 mg/kg ip; 24 hrs 1.0 mg/kg ip.

REFERENCES FOR EXAMPLE 38

-   Cechetto D F, Wilson J X, Smith K E, Wolski D, Silver M D, Hachinski    V C (1989). Autonomic and myocardial changes in middle cerebral    artery occlusion: stroke models in the rat. Brain Res 502:296-305.-   Kolb E, Sutherland R J, Whishaw I Q (1983). A comparison of the    contributions of the frontal and parietal association cortex to    spatial localizations in the rat. Behav. Neurosci. 97:13-27.-   Menzies S A, Hoff J T, Betz A L (1992). Middle cerebral artery    occlusion in rats: A neurological and pathological evaluation of a    reproducible model. Neurosurgery 31:100-106.-   Sauer D, Allegrini P R, Cosenti A, Pataki A, Amaceker X, Fagg G E    (1993). Characterization of the cerebroprotective efficacy of the    competitive NMDA receptor antagonist CGP40116 in a rat model of    focal cerebral ischemia: An in vivo magnetic resonance imaging    study. J. Cerebr. Blood Flow and Metabol. 13:595-602.-   Stephanovich C, Editor, Stroke: Animal Models, Pergamon Press, 1983-   Tamura A, Graham D I, McCulloch J, Teasdale G H (1981). Focal    cerebral ischemia in the rat: 1. Description of technique and early    neuropathological consequences following MCA occlusion. J. Cereb.    Blood Flow and Metab. 1:53-60.-   Teasdale G, Tyson G, Tamura A, Graham D I, McCulloch J. Focal    cerebral ischaemia in the rat: Neuropatholgy, local cerebral blood    flow and cerebrovascular permeability. In Stroke: Animal Models,    Stephanovic C, Editor, Pergamon Press 1983, pp. 83-97.-   Yamamoto M, Tamura A, Kirino T, Shimitzu M, Sano K (1966).    Behavioral changes after focal cerebral ischemia by left middle    cerebral artery occlusion in rats. Brain Re. 452:323-328.-   Yamori Y et al. (1976). Pathogenic similarity of strokes in stroke    prone spontaneously hypertensive rats and humans. Stroke 7:46-53.-   Young W. DeCrescito V, Flamm E S, Hadani M, Rappaport H, Cornu P    (1986), Tissue Na, K, and Ca changes in regional cerebral ischemia:    Their measurement and interpretation. Central Nervous System Trauma,    3:215-234.

1-41. (canceled)
 42. A method of treating a subject afflicted withdementia which comprises administering to the subject a pharmaceuticalcomposition consisting of an amount of the mesylate salt ofR(+)-N-propargyl-1-aminoindan effective to treat the subject and atleast one pharmaceutically acceptable carrier.
 43. The method of claim42, wherein the dementia is of the Alzheimer type.
 44. The method ofclaim 42, wherein the subject is human and the effective amount is fromabout 0.1 mg to about 100 mg per day.
 45. The method of claim 44,wherein the effective amount is from about 1 mg to about 10 mg per day.46. The method of claim 45, wherein the effective amount is 1 mg perday.
 47. The method of claim 42, wherein the pharmaceutical compositionis administered orally, rectally, intravenously, transdermally, orparenterally.
 48. The method of claim 47, wherein the pharmaceuticalcomposition is administered orally.
 49. The method of claim 46, whereinpharmaceutical composition is administered orally, rectally,intravenously, transdermally, or parenterally.
 50. The method of claim49, wherein the pharmaceutical composition is administered orally.
 51. Amethod of treating a subject afflicted with dementia which comprisesadministering to the subject a pharmaceutical composition consistingessentially of an amount of the mesylate salt ofR(+)-N-propargyl-1-aminoindan effective to treat the subject and atleast one pharmaceutically acceptable carrier.
 52. The method of claims51, wherein the dementia is of the Alzheimer type.
 53. The method ofclaim 51, wherein the subject is human and the effective amount is fromabout 0.1 mg to about 100 mg per day.
 54. The method of claim 53,wherein the effective amount is from about 1 mg to about 10 mg per day.55. The method of claim 54, wherein the effective amount is 1 mg perday.
 56. The method of claim 51, wherein the pharmaceutical compositionis administered orally, rectally, intravenously, transdermally, orparenterally.
 57. The method of claim 56, wherein the pharmaceuticalcomposition is administered orally.
 58. The method of claim 55, whereinthe pharmaceutical composition is administered orally, rectally,intravenously, transdermally, or parenterally.
 59. The method of claim58, wherein the pharmaceutical composition is administered orally.
 60. Amethod of treating a subject afflicted with dementia which comprisesadministering to the subject a pharmaceutical composition comprising anamount of the mesylate salt of R(+)-N-propargyl-1-aminoindan effectiveto treat the subject and at least one pharmaceutically acceptablecarrier.
 61. The method of claims 60, wherein the dementia is of theAlzheimer type.
 62. The method of claim 60, wherein the subject is humanand the effective amount is from about 0.1 mg to about 100 mg per day.63. The method of claim 62, wherein the effective amount is from about 1mg to about 10 mg per day.
 64. The method of claim 63, wherein theeffective amount is 1 mg per day.
 65. The method of claim 60, whereinthe pharmaceutical composition is administered orally, rectally,intravenously, transdermally, or parenterally.
 66. The method of claim65, wherein the pharmaceutical composition is administered orally. 67.The method of claim 64, wherein the pharmaceutical composition isadministered orally, rectally, intravenously, transdermally, orparenterally.
 68. The method of claim 67, wherein the pharmaceuticalcomposition is administered orally.